Methods and compositions for use in genome modification in plants

ABSTRACT

The present disclosure is in the field of plant transformation. The disclosure provides methods for increasing the rate of site-directed integration of a sequence of interest in plants. The disclosure also provides methods for increasing the efficiency of Rhizobiales-mediated plant transformation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/311,749, which was filed in the U.S. on Dec. 20, 2018 as a U.S.National Stage Application of International Application No.PCT/US2017/039502, which was filed internationally on Jun. 27, 2017,which claims priority to U.S. Provisional Patent Application No.62/355,715, filed Jun. 28, 2016, all of which are incorporated byreference in their entireties herein.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(777052057501SEQLIST.xml; Size: 29,187 bytes; and Date of Creation: Jun.1, 2023) is herein incorporated by reference in its entirety.

FIELD

This disclosure relates to the field of plant transformation. Methodsof, and compositions for, transfer of T-strands of DNA fromAgrobacterium plasmids are provided. Also provided are methods of, andcompositions for, using Agrobacterium-mediated transfer of T-strands ofDNA to promote templated gene editing and site-directed integration oftransgenes. Also provided are methods for increasing the efficiency ofAgrobacterium-mediated transformation. Non-Agrobacterium-mediatedtransformation methods and compositions are also provided.

BACKGROUND

Agrobacterium-mediated transformation utilizes Agrobacterium tumefaciensto transfer single-stranded DNA synthesized from recombinant plasmids toplant cells. Transformation of plant cells often requires co-culturingwith A. tumefaciens. A DNA sequence of interest is incorporated intospecially-constructed DNA plasmids where the DNA sequence of interest isflanked by an Agrobacterium tumor-inducing (Ti) plasmid right border DNAregion and a left border DNA region. The Agrobacterium-mediatedtransformation process initiates when an endonuclease, VirD2, nicks theDNA plasmid at the right border and left border regions to release asingle-stranded transfer DNA (also called the T-strand). The T-strandtransfers transgenes situated between the right and left borders intothe targeted plant cells, where the T-strand can integrate into thegenome (see, for example, U.S. Pat. No. 5,034,322; Pitzschke and Hirt.The EMBO Journal (2010) 29: 1021-1032). The right border, but not theleft border, is essential for successful transformation of plant cells(see Wang et al. Cell (1984) 38:455-462; and van Haaren et al. PlantMolecular Biology (1987) 8: 95-104). Plant cells that have beentransformed via Agrobacterium-mediated transformation can be manipulatedto regenerate into a whole, fertile plant.

Successful Agrobacterium-mediated transformation of a plant celltypically results in a random integration in the plant genome. Suchrandom integrations can have deleterious effects to the plant cell ifthe transgene inserts into an essential endogenous gene. Similarly, suchrandom integrations can have a deleterious effect on expression of thetransgene due to position effects of the chromosome where the transgeneinserted. There exists a need in the art for an Agrobacterium-mediatedtransformation method for plant cells that promotes templated geneediting and site-directed integration of transgenes.

SUMMARY

In one aspect, the present disclosure provides a method of transforminga plant cell, comprising contacting the plant cell with a Rhizobialescell capable of transforming the plant cell, where the Rhizobiales cellcomprises at least one vector capable of forming two T-strands that areessentially complementary in at least a portion of the T-strands. In afurther aspect, the at least one vector comprises a first right borderDNA sequence (RB1), a second right border DNA sequence (RB2), and atleast one sequence of interest, and the RB1 is positioned in the vectorto initiate synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the RB2 is positioned in the vector to initiate synthesisof a second T-strand such that the sequence of interest is in theanti-sense orientation relative to the sequence of interest in the firstT-strand, and the two T-strands resulting from initiation at RB1 and RB2are essentially complementary in at least a portion of the sequence ofinterest. In an alternative further aspect, the at least one vectorcomprises a RB1, a RB2, a sequence of interest, a first left border DNAsequence (LB1) and a second left border DNA sequence (LB2), where thevector is configured such that the RB1 is paired with the LB1 which arepositioned in the vector to initiate (RB1) and terminate (LB1) synthesisof a first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the RB2 ispaired with the LB2 which are positioned in the vector to initiate (RB2)and terminate (LB2) synthesis of a second T-strand such that thesequence of interest is in an anti-sense orientation relative to thesequence of interest in the first T-strand, and where the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryin at least a portion of the sequence of interest. In anotheralternative further aspect, the vector comprises a first sequence ofinterest and a second sequence of interest, where the first sequence ofinterest is essentially identical to the second sequence of interest,where the vector further comprises a RB1 and a LB1 which are positionedin the vector to initiate (RB1) and terminate (LB1) synthesis of a firstT-strand such that the first sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand, and the vectorfurther comprises a RB2 and a LB2 which are positioned in the vector toinitiate (RB2) and terminate (LB2) synthesis of a second T-strand suchthat the second sequence of interest is in an anti-sense orientationrelative to the first sequence of interest in the first T-strand, andwhere the two T-strands resulting from initiation at RB1 and RB2 areessentially complementary in at least a portion of the first sequence ofinterest and the second sequence of interest. In yet another alternativefurther aspect, the Rhizobiales cell comprises at least a first vectorand a second vector, where each vector comprises essentially identicalsequences of interest, and where the first vector comprises a RB1 and aLB1 which are positioned in the first vector to initiate (RB1) andterminate (LB1) synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and where the second vector comprises a RB2 and a LB2) whichare positioned in the second vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the sequence of interestis in an anti-sense orientation relative to the sequence of interest inthe first T-strand, and where the two T-strands resulting frominitiation at RB1 and RB2 are essentially complementary in at least aportion of the sequence of interest. In another alternative furtheraspect, the vector comprises a first sequence of interest, a secondsequence of interest different from the first sequence of interest, atleast two RB DNA sequences, and one or more optional LB DNA sequences,wherein the first RB DNA sequence (RB1) and a first LB DNA sequence(LB1) are positioned in the vector to initiate (RB1) and terminate (LB1)synthesis of the first T-strand such that the first sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the vector configuration further comprises a second RB DNA sequence(RB2) and a second LB DNA sequence (LB2) which are positioned in thevector to initiate (RB2) and terminate (LB2) synthesis of a secondT-strand such that the first sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand, wherein thetwo T-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other in at least a portion of the first sequenceof interest, and wherein the vector configuration further comprises athird RB DNA sequence (RB3) and a third LB DNA sequence (LB3) which arepositioned in the vector to initiate (RB3) and terminate (LB3) synthesisof a third T-strand such that the second sequence of interest is in thesense orientation from the 5′ to 3′ end of the third T-strand; and thevector configuration further comprises a fourth RB DNA sequence (RB4)and a fourth LB DNA sequence (LB4) which are positioned in the vector toinitiate (RB4) and terminate (LB4) synthesis of a fourth T-strand suchthat the second sequence of interest is in an anti-sense orientationfrom the 5′ to 3′ end of the fourth T-strand, and the two T-strandsresulting from initiation at RB3 and RB4 are essentially complementaryto each other in at least a portion of the second sequence of interest.In some embodiments, RB1 is 5′ to LB2. In some embodiments, RB1 is 3′ toLB2. In some embodiments, RB2 is 5′ to LB1. In some embodiments, RB2 is3′ to LB1.

In one aspect, the instant disclosure provides a method of transforminga plant cell, comprising contacting the plant cell with two or moreRhizobiales cells capable of transforming the plant cell, where the twoor more Rhizobiales cells each contain one of at least two vectorscapable of forming two essentially complementary T-strands. In a furtheraspect, each vector comprises an essentially identical sequence ofinterest, and where the first vector comprises a first right border DNAsequence (RB1), and where the RB1 is positioned in the vector toinitiate synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the second vector comprises a second right border DNAsequence (RB2) which is positioned in the vector to initiate synthesisof a second T-strand such that the sequence of interest is in theanti-sense orientation relative to the sequence of interest in the firstT-strand, and where the two T-strands resulting from initiation at RB1and RB2 are essentially complementary in at least a portion of thesequence of interest. In an alternative further aspect, each vectorcomprises an essentially identical sequence of interest, and where thefirst vector comprises a RB1 and a first left border DNA sequence (LB1)which are positioned in the first vector to initiate (RB1) and terminate(LB1) synthesis of a first T-strand such that the sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the second vector comprises a RB2 and a second left border DNAsequence (LB2) which are positioned in the second vector to initiate(RB2) and terminate (LB2) synthesis of a second T-strand such that thesequence of interest is in an anti-sense orientation from the 5′ to 3′end of the second T-strand, and where the sequence of interest in thetwo T-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other. In some embodiments, RB1 is 5′ to LB2. Insome embodiments, RB1 is 3′ to LB2. In some embodiments, RB2 is 5′ toLB1. In some embodiments, RB2 is 3′ to LB1.

In one aspect, the instant disclosure provides a method of increasingthe rate of site directed integration of a sequence of interest,comprising contacting a plant cell with at least one vector capable offorming two essentially complementary T-strands. In a further aspect,the at least one vector comprises a first right border DNA sequence(RB1), a second right border DNA sequence (RB2), and at least onesequence of interest, where the RB1 is positioned in the vector toinitiate synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the RB2 is positioned in the vector to initiate synthesisof a second T-strand such that the sequence of interest is in theanti-sense orientation relative to the sequence of interest in the firstT-strand, and where the two T-strands resulting from initiation at RB1and RB2 are essentially complementary in at least a portion of thesequence of interest. In an alternative further aspect, the at least onevector comprises a RB1, a RB2, a sequence of interest, a first leftborder DNA sequence (LB1), a second left border DNA sequence (LB2) andwhere the vector is configured such that the RB1 is paired with the LB1which are positioned in the vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andthe RB2 is paired with the LB2 which are positioned in the vector toinitiate (RB2) and terminate (LB2) synthesis of a second T-strand suchthat the sequence of interest is in an anti-sense orientation relativeto the sequence of interest in the first T-strand, and where thesequence of interest in the two T-strands resulting from initiation atRB1 and RB2 are essentially complementary in at least a portion of thesequence of interest. In another alternative further aspect, the vectorcomprises a first sequence of interest and a second sequence ofinterest, where the first sequence of interest is essentially identicalto the second sequence of interest; where the vector further comprises aRB1 and a LB1 which are positioned in the vector to initiate (RB1) andterminate (LB1) synthesis of a first T-strand such that the firstsequence of interest is in the sense orientation from the 5′ to 3′ endof the first T-strand; and the vector further comprises a RB2 and a LB2which are positioned in the vector to initiate (RB2) and terminate (LB2)synthesis of a second T-strand such that the second sequence of interestis in an anti-sense orientation relative to the first sequence ofinterest in the first T-strand, and where the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the first sequence of interest and the second sequence ofinterest. In yet another alternative further aspect, the Rhizobialescell comprises at least a first and a second vector, where each vectorcomprises essentially identical sequences of interest, and where thefirst vector comprises a RB1 and a LB1 which are positioned in the firstvector to initiate (RB1) and terminate (LB1) synthesis of a firstT-strand such that the sequence of interest is in the sense orientationfrom the 5′ to 3′ end of the first T-strand; and the second vectorcomprises a RB2 and a LB2 which are positioned in the second vector toinitiate (RB2) and terminate (LB2) synthesis of a second T-strand suchthat the sequence of interest is in an anti-sense orientation relativeto the sequence of interest in the first T-strand, and where the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest. Inanother alternative further aspect, the vector comprises a firstsequence of interest, a second sequence of interest different from thefirst sequence of interest, at least two RB DNA sequences, and one ormore optional LB DNA sequences, wherein the first RB DNA sequence (RB1)and a first LB DNA sequence (LB1) are positioned in the vector toinitiate (RB1) and terminate (LB1) synthesis of the first T-strand suchthat the first sequence of interest is in the sense orientation from the5′ to 3′ end of the first T-strand; and the vector configuration furthercomprises a second RB DNA sequence (RB2) and a second LB DNA sequence(LB2) which are positioned in the vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the first sequence ofinterest is in an anti-sense orientation from the 5′ to 3′ end of thesecond T-strand, wherein the two T-strands resulting from initiation atRB1 and RB2 are essentially complementary to each other in at least aportion of the first sequence of interest, and wherein the vectorconfiguration further comprises a third RB DNA sequence (RB3) and athird LB DNA sequence (LB3) which are positioned in the vector toinitiate (RB3) and terminate (LB3) synthesis of a third T-strand suchthat the second sequence of interest is in the sense orientation fromthe 5′ to 3′ end of the third T-strand; and the vector configurationfurther comprises a fourth RB DNA sequence (RB4) and a fourth LB DNAsequence (LB4) which are positioned in the vector to initiate (RB4) andterminate (LB4) synthesis of a fourth T-strand such that the secondsequence of interest is in an anti-sense orientation from the 5′ to 3′end of the fourth T-strand, and the two T-strands resulting frominitiation at RB3 and RB4 are essentially complementary to each other inat least a portion of the second sequence of interest. In someembodiments, RB1 is 5′ to LB2. In some embodiments, RB1 is 3′ to LB2. Insome embodiments, RB2 is 5′ to LB1. In some embodiments, RB2 is 3′ toLB1.

In one aspect, the instant disclosure provides a method of increasingthe rate of site directed integration of a sequence of interest,comprising contacting a plant cell with two or more Rhizobiales cells,where the two or more Rhizobiales cells each contain one of at least twovectors capable of forming two essentially complementary T-strands. In afurther aspect, each vector comprises an essentially identical sequenceof interest, and where the first vector comprises a first right borderDNA sequence (RB1), and where the RB1 is positioned in the vector toinitiate synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the second vector comprises a second right border DNAsequence (RB2) which is positioned in the vector to initiate synthesisof a second T-strand such that the sequence of interest is in theanti-sense orientation relative to the sequence of interest in the firstT-strand, and where the two T-strands resulting from initiation at RB1and RB2 are essentially complementary in at least a portion of thesequence of interest. In an alternative further aspect, each vectorcomprises an essentially identical sequence of interest, and where thefirst vector comprises a RB1 and a first left border (LB1) which arepositioned in the first vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andthe second vector comprises a RB2 and a second left border (LB2) whichare positioned in the second vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the sequence of interestis in an anti-sense orientation from the 5′ to 3′ end of the secondT-strand, and where the sequence of interest in the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryto each other. In some embodiments, the Rhizobiales cell is selectedfrom an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp.,an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and aSinorhizobium spp. In some embodiments, the Rhizobiales cell furthercontains a vector comprising at least one expression cassette, whereinthe expression cassettes comprise a sequence encoding a protein involvedin DNA repair, and wherein the protein is selected from the groupcomprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof. In some embodiments,RB1 is 5′ to LB2. In some embodiments, RB1 is 3′ to LB2. In someembodiments, RB2 is 5′ to LB1. In some embodiments, RB2 is 3′ to LB1.

In one aspect, the instant disclosure provides a method of transforminga plant cell, comprising contacting the plant cell with a Rhizobialescell capable of transforming the plant cell, where the Rhizobiales cellcomprises at least one vector comprising a right border DNA sequence(RB) and a left border DNA sequence (LB) and where the vector comprisesbetween the RB and LB: (i) a first sequence of interest in a senseorientation relative to the RB, (ii) a spacer, and (iii) a secondsequence of interest in an anti-sense orientation relative to the RB,where the first sequence of interest and second sequence of interest areessentially complementary and after synthesis of the T-strand anneal toform a double-stranded DNA. In some embodiments, the Rhizobiales cell isselected from an Agrobacterium spp., a Bradyrhizobium spp., aMesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., aRhizobium spp., and a Sinorhizobium spp. In some embodiments, theRhizobiales cell further contains a vector comprising at least oneexpression cassette, wherein the expression cassettes comprise asequence encoding a protein involved in DNA repair, and wherein theprotein is selected from the group comprising a vir gene from the Tiplasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or anycombination thereof.

In one aspect, the instant disclosure provides a method of increasingthe rate of site directed integration of a double-stranded DNA into agenome of a plant cell, comprising contacting the plant cell with atleast one vector comprising a right border DNA sequence (RB) and a leftborder DNA sequence (LB), where the vector comprises between the RB andLB: (i) a first sequence of interest in a sense orientation relative tothe RB, (ii) a spacer, and (iii) a second sequence of interest in ananti-sense orientation relative to the RB, where the first sequence ofinterest and second sequence of interest are essentially complementaryand after synthesis of the T-anneal, where the double-stranded DNA isintegrated into a genome of a plant cell.

In one aspect, the instant disclosure provides an Agrobacterium cellcomprising at least one vector that is capable of forming twoessentially complementary T-strands.

In one aspect, the instant disclosure provides an Agrobacterium cellcomprising at least one vector comprising a right border DNA sequence(RB) and a left border DNA sequence (LB) and where the vector comprisesbetween the RB and LB: (i) a first sequence of interest in a senseorientation relative to the RB, (ii) a spacer, and (iii) a secondsequence of interest in an anti-sense orientation relative to the RB,where the first sequence of interest and second sequence of interest areessentially complementary, and after synthesis of the T-strand anneal toform a double-stranded DNA.

In one aspect, the instant disclosure provides a method of transforminga plant genome, comprising contacting at least one plant cell on aco-culture medium for at least 2 days, with at least one Rhizobialescell capable of transforming the plant cell, where the Rhizobiales cellcomprises at least one vector capable of forming two essentiallycomplementary T-strands. In some embodiments, the Rhizobiales cell isselected from an Agrobacterium spp., a Bradyrhizobium spp., aMesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., aRhizobium spp., and a Sinorhizobium spp. In some embodiments, theRhizobiales cell further contains a vector comprising at least oneexpression cassette, wherein the expression cassettes comprise asequence encoding a protein involved in DNA repair, and wherein theprotein is selected from the group comprising a vir gene from the Tiplasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or anycombination thereof.

In one aspect, the instant disclosure provides a method of transforminga plant genome, comprising contacting at least one plant cell on aco-culture medium for at least 2 days, with at least one Rhizobialescell capable of transforming the plant cell, where the Rhizobiales cellcomprises at least one vector comprising a right border (RB) DNAsequence and a left border (LB) DNA sequence and where the vectorfurther comprises between the RB and LB DNA sequences: (i) a firstsequence of interest in a sense orientation relative to the RB DNAsequence, and (ii) a spacer, and (iii) a second sequence of interest inan anti-sense orientation relative to the RB DNA sequence, where thefirst sequence of interest and second sequence of interest areessentially complementary, and after synthesis of the T-strand anneal toform a double-stranded DNA. In some embodiments, the Rhizobiales cell isselected from an Agrobacterium spp., a Bradyrhizobium spp., aMesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., aRhizobium spp., and a Sinorhizobium spp. In some embodiments, theRhizobiales cell further contains a vector comprising at least oneexpression cassette, wherein the expression cassettes comprise asequence encoding a protein involved in DNA repair, and wherein theprotein is selected from the group comprising a vir gene from the Tiplasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or anycombination thereof.

In one aspect, the instant disclosure provides a method of transforminga plant genome, comprising contacting at least one plant cell on aco-culture medium for at least 3 days, with at least one Rhizobialescell capable of transforming the plant cell, where the Rhizobiales cellcomprises at least one vector capable of forming two essentiallycomplementary T-strands. In some embodiments, the Rhizobiales cell isselected from an Agrobacterium spp., a Bradyrhizobium spp., aMesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., aRhizobium spp., and a Sinorhizobium spp. In some embodiments, theRhizobiales cell further contains a vector comprising at least oneexpression cassette, wherein the expression cassettes comprise asequence encoding a protein involved in DNA repair, and wherein theprotein is selected from the group comprising a vir gene from the Tiplasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or anycombination thereof.

In one aspect, the instant disclosure provides a method of transforminga plant genome, comprising contacting at least one plant cell on aco-culture medium for at least 3 days, with at least one Rhizobialescell capable of transforming the plant cell, where the Rhizobiales cellcomprises at least one vector comprising a right border (RB) DNAsequence and a left border (LB) DNA sequence and where the vectorfurther comprises between the RB and LB DNA sequences: (i) a firstsequence of interest in a sense orientation relative to the RB DNAsequence, and (ii) a spacer, and (iii) a second sequence of interest inan anti-sense orientation relative to the RB DNA sequence, where thefirst sequence of interest and second sequence of interest areessentially complementary, and after synthesis of the T-strand anneal toform a double-stranded DNA. In some embodiments, the Rhizobiales cell isselected from an Agrobacterium spp., a Bradyrhizobium spp., aMesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., aRhizobium spp., and a Sinorhizobium spp. In some embodiments, theRhizobiales cell further contains a vector comprising at least oneexpression cassette, wherein the expression cassettes comprise asequence encoding a protein involved in DNA repair, and wherein theprotein is selected from the group comprising a vir gene from the Tiplasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or anycombination thereof.

Several embodiments relate to a method of providing a sequence ofinterest to a plant cell, comprising contacting the plant cell with aRhizobiales cell capable of transforming the plant cell, wherein theRhizobiales cell comprises at least one vector capable of forming twoT-strands that are essentially complementary in at least a portion ofthe T-strands, wherein the at least one vector comprises a first rightborder DNA sequence (RB1), a second right border DNA sequence (RB2), andat least one sequence of interest, and wherein the RB1 is positioned inthe vector to initiate synthesis of a first T-strand such that thesequence of interest is in the sense orientation from the 5′ to 3′ endof the first T-strand; and the RB2 is positioned in the vector toinitiate synthesis of a second T-strand such that the sequence ofinterest is in the anti-sense orientation relative to the sequence ofinterest in the first T-strand, and wherein the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the sequence of interest. In some embodiments, the RB1 andthe RB2 are essentially homologous. In some embodiments, the LB1 and theLB2 are essentially homologous. In some embodiments, the RB1 and the RB2are not essentially homologous. In some embodiments, the LB1 and LB2 arenot essentially homologous. In some embodiments, at least one of the RB1and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Flp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell. In some embodiments, the Rhizobiales cell is selected from anAgrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., anOchrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and aSinorhizobium spp. In some embodiments, the Rhizobiales cell furthercontains a vector comprising at least one expression cassette, whereinthe expression cassettes comprise a sequence encoding a protein involvedin DNA repair, and wherein the protein is selected from the groupcomprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof.

A method of providing a sequence of interest to a plant cell, comprisingcontacting the plant cell with a Rhizobiales cell capable oftransforming the plant cell, wherein the Rhizobiales cell comprises atleast one vector capable of forming two T-strands that are essentiallycomplementary in at least a portion of the T-strands, wherein the atleast one vector comprises a RB1, a RB2, a sequence of interest, a firstleft border DNA sequence (LB1) and a second left border DNA sequence(LB2), wherein the vector is configured such that the RB1 is paired withthe LB1 which are positioned in the vector to initiate (RB1) andterminate (LB1) synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the RB2 is paired with the LB2 which are positioned in thevector to initiate (RB2) and terminate (LB2) synthesis of a secondT-strand such that the sequence of interest is in an anti-senseorientation relative to the sequence of interest in the first T-strand,and wherein the two T-strands resulting from initiation at RB1 and RB2are essentially complementary in at least a portion of the sequence ofinterest. In some embodiments, the RB1 and the RB2 are essentiallyhomologous. In some embodiments, the LB1 and the LB2 are essentiallyhomologous. In some embodiments, the RB1 and the RB2 are not essentiallyhomologous. In some embodiments, the LB1 and LB2 are not essentiallyhomologous. In some embodiments, at least one of the RB1 and RB2comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. Insome embodiments, the Rhizobiales cell further contains a vectorcomprising at least one expression cassette, wherein the expressioncassettes comprise a sequence encoding a protein involved in DNA repair,and wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, the plant cell alreadycomprises a site-specific enzyme. In some embodiments, the plant cell isselected from the group consisting of a corn cell, a soybean cell, acanola cell, a cotton cell, a wheat cell, or a sugarcane cell. In someembodiments, a nucleotide sequence encoding the site-specific enzyme isstably transformed into the plant cell.

A method of providing a sequence of interest to a plant cell, comprisingcontacting the plant cell with a Rhizobiales cell capable oftransforming the plant cell, wherein the Rhizobiales cell comprises atleast one vector capable of forming two T-strands that are essentiallycomplementary in at least a portion of the T-strands, wherein the vectorcomprises a first sequence of interest and a second sequence ofinterest, wherein the first sequence of interest is essentiallyidentical to the second sequence of interest; wherein the vector furthercomprises a RB1 and a LB1 which are positioned in the vector to initiate(RB1) and terminate (LB1) synthesis of a first T-strand such that thefirst sequence of interest is in the sense orientation from the 5′ to 3′end of the first T-strand; and the vector further comprises a RB2 and aLB2 which are positioned in the vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the second sequence ofinterest is in an anti-sense orientation relative to the first sequenceof interest in the first T-strand, and wherein the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryin at least a portion of the first sequence of interest and the secondsequence of interest. In some embodiments, the RB1 and the RB2 areessentially homologous. In some embodiments, the LB1 and the LB2 areessentially homologous. In some embodiments, the RB1 and the RB2 are notessentially homologous. In some embodiments, the LB1 and LB2 are notessentially homologous. In some embodiments, at least one of the RB1 andRB2 comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell. In some embodiments, the Rhizobiales cell is selected from anAgrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., anOchrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and aSinorhizobium spp. In some embodiments, the Rhizobiales cell furthercontains a vector comprising at least one expression cassette, whereinthe expression cassettes comprise a sequence encoding a protein involvedin DNA repair, and wherein the protein is selected from the groupcomprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof.

A method of providing a sequence of interest to a plant cell, comprisingcontacting the plant cell with a Rhizobiales cell capable oftransforming the plant cell, wherein the Rhizobiales cell comprises atleast one vector capable of forming two T-strands that are essentiallycomplementary in at least a portion of the T-strands, wherein theRhizobiales cell comprises at least a first vector and a second vector,wherein each vector comprises essentially identical sequences ofinterest, and wherein the first vector comprises a RB1 and a LB1 whichare positioned in the first vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andwherein the second vector comprises a RB2 and a LB2) which arepositioned in the second vector to initiate (RB2) and terminate (LB2)synthesis of a second T-strand such that the sequence of interest is inan anti-sense orientation relative to the sequence of interest in thefirst T-strand, and wherein the two T-strands resulting from initiationat RB1 and RB2 are essentially complementary in at least a portion ofthe sequence of interest. In some embodiments, the RB1 and the RB2 areessentially homologous. In some embodiments, the LB1 and the LB2 areessentially homologous. In some embodiments, the RB1 and the RB2 are notessentially homologous. In some embodiments, the LB1 and LB2 are notessentially homologous. In some embodiments, at least one of the RB1 andRB2 comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell. In some embodiments, the Rhizobiales cell is selected from anAgrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., anOchrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and aSinorhizobium spp. In some embodiments, the Rhizobiales cell furthercontains a vector comprising at least one expression cassette, whereinthe expression cassettes comprise a sequence encoding a protein involvedin DNA repair, and wherein the protein is selected from the groupcomprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof.

A method of providing a sequence of interest to a plant cell, comprisingcontacting the plant cell with a Rhizobiales cell capable oftransforming the plant cell, wherein the Rhizobiales cell comprises atleast one vector capable of forming two T-strands that are essentiallycomplementary in at least a portion of the T-strands, wherein the atleast one vector comprises a first sequence of interest, a secondsequence of interest different from the first sequence of interest, atleast two RB DNA sequences, and one or more optional LB DNA sequences,wherein the first RB DNA sequence (RB1) and a first LB DNA sequence(LB1) are positioned in the vector to initiate (RB1) and terminate (LB1)synthesis of the first T-strand such that the first sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the vector configuration further comprises a second RB DNA sequence(RB2) and a second LB DNA sequence (LB2) which are positioned in thevector to initiate (RB2) and terminate (LB2) synthesis of a secondT-strand such that the first sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand, wherein thetwo T-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other in at least a portion of the first sequenceof interest, and wherein the vector configuration further comprises athird RB DNA sequence (RB3) and a third LB DNA sequence (LB3) which arepositioned in the vector to initiate (RB3) and terminate (LB3) synthesisof a third T-strand such that the second sequence of interest is in thesense orientation from the 5′ to 3′ end of the third T-strand; and thevector configuration further comprises a fourth RB DNA sequence (RB4)and a fourth LB DNA sequence (LB4) which are positioned in the vector toinitiate (RB4) and terminate (LB4) synthesis of a fourth T-strand suchthat the second sequence of interest is in an anti-sense orientationfrom the 5′ to 3′ end of the fourth T-strand, and the two T-strandsresulting from initiation at RB3 and RB4 are essentially complementaryto each other in at least a portion of the second sequence of interest.In some embodiments, the RB1 and the RB2 are essentially homologous. Insome embodiments, the LB1 and the LB2 are essentially homologous. Insome embodiments, the RB1 and the RB2 are not essentially homologous. Insome embodiments, the LB1 and LB2 are not essentially homologous. Insome embodiments, at least one of the RB1 and RB2 comprise anAgrobacterium Ti plasmid right border consensus DNA sequence. In someembodiments, the right border consensus DNA sequence is selected fromSEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of theRB1 and RB2 comprise a sequence selected from SEQ ID NOs: 1-13. In someembodiments, at least one of the RB1 and RB2 comprise a sequence atleast 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ IDNO:12. In some embodiments, at least one of the LB1 and LB2 comprise anAgrobacterium Ti plasmid left border consensus DNA sequence. In someembodiments, the left border consensus DNA sequence is selected from SEQID NO: 23 or SEQ ID NO: 24. In some embodiments, at least one of the LB1and LB2 comprise a sequence selected from SEQ ID NOs: 14-20. In someembodiments, at least one of the LB1 and LB2 comprise a sequence atleast 80% identical to SEQ ID NO:19. In some embodiments, the sequenceof interest comprises one or more expression cassettes. In someembodiments, the sequence of interest comprises one or more sequencesselected from: a gene, a portion of a gene, an intergenic sequence, anenhancer, a promoter, an intron, an exon, a sequence encoding atranscription termination sequence, a sequence encoding a chloroplasttargeting peptide, a sequence encoding a mitochondrial targetingpeptide, an insulator sequence, a sequence encoding an anti-sense RNAconstruct, a sequence encoding non-protein-coding RNA (npcRNA), asequence encoding a recombinase, a sequence encoding a recombinaserecognition site, a landing pad, an editing template, an expressioncassette, a stack of two or more expression cassettes encodingtransgenes, a sequence encoding a site-specific enzyme, a sequenceencoding a site-specific enzyme target site, a sequence encoding aselection marker, a sequence encoding a cell factor that functions toincrease DNA repair, a sequence comprising a linker or a spacer, asequence comprising one or more restriction enzyme sites, a sequence fortemplated genome editing, and any combination thereof. In someembodiments, the sequence of interest comprises a sequence encoding asite-specific enzyme target site 5′ to an expression cassette and asequence encoding a site-specific enzyme target site 3′ to an expressioncassette. In some embodiments, the sequence of interest does notcomprise a homology arm DNA sequence. In some embodiments, the sequenceof interest comprises at least one homology arm DNA sequence. In someembodiments, the sequence of interest comprises both a left homology armDNA sequence and a right homology arm DNA sequence. In some embodiments,the sequence of interest comprises i) a first sequence positionedbetween the left homology arm DNA sequence and the right homology DNAarm sequence, and ii) a second sequence that is not positioned betweenthe left homology arm DNA sequence and the right homology arm DNAsequence. In some embodiments, the sequence of interest comprises asequence positioned between the left homology arm DNA sequence and theright homology DNA arm sequence, where the left and right homology armsequences are positioned between two sequences encoding site-specificenzyme target sites. In some embodiments, the sequence of interestcomprises i) a first sequence positioned between the left homology armDNA sequence and the right homology DNA arm sequence where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites, and ii) a second sequencethat is not positioned between sequences encoding site-specific enzymetarget sites. In some embodiments, the sequence of interest comprises i)a first sequence positioned between the left homology arm DNA sequenceand the right homology DNA arm sequence where the left and righthomology arm sequences are positioned between two sequences encodingsite-specific enzyme target sites, and ii) a second sequence encodingthe site-specific enzyme, where the second sequence is not positionedbetween sequences encoding site-specific enzyme target sites. In someembodiments, at least one homology arm DNA sequence comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to a target sequence in the plant genome. In someembodiments, the target sequence in the plant genome is a genicsequence. In some embodiments, the target sequence in the plant genomeis a non-genic sequence. In some embodiments, the sequence of interestflanked by homology arms comprises a sequence that is at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to a native gene of the plant cell. In someembodiments, the sequence of interest comprises a protein-codingsequence. In some embodiments, the sequence of interest comprises anon-protein-coding RNA. In some embodiments, the non-protein-coding RNAis selected from the group consisting of: a microRNA (miRNA), a miRNAprecursor, a small interfering RNA (siRNA), a small RNA (22-26 nt inlength) and precursor encoding same, a heterochromatic siRNA (hc-siRNA),a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpindsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisensesiRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and asingle-guide RNA (sgRNA), or any combination thereof. In someembodiments, the site-specific enzyme is selected from a groupconsisting of an endonuclease, a recombinase, and a transposase. In someembodiments, the endonuclease is selected from a meganuclease, a zincfinger nuclease, a transcription activator-like effector nuclease(TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasYnuclease, and a Cpf1 nuclease. In some embodiments, the Cas9 nuclease isselected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1,Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. Insome embodiments, the Rhizobiales cell further contains a vectorcomprising at least one expression cassette, wherein the expressioncassettes comprise a sequence encoding a protein involved in DNA repair,and wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, the plant cell alreadycomprises a site-specific enzyme. In some embodiments, the plant cell isselected from the group consisting of a corn cell, a soybean cell, acanola cell, a cotton cell, a wheat cell, or a sugarcane cell. In someembodiments, a nucleotide sequence encoding the site-specific enzyme isstably transformed into the plant cell.

Several embodiments relate to a method of transforming a plant cell,comprising contacting the plant cell with two or more Rhizobiales cellscapable of transforming the plant cell, wherein the two or moreRhizobiales cells each contain one of at least two vectors capable offorming two essentially complementary T-strands, wherein each vectorcomprises an essentially identical sequence of interest, and where thefirst vector comprises a first right border DNA sequence (RB1), andwherein the RB1 is positioned in the vector to initiate synthesis of afirst T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the secondvector comprises a second right border DNA sequence (RB2) which ispositioned in the vector to initiate synthesis of a second T-strand suchthat the sequence of interest is in the anti-sense orientation relativeto the sequence of interest in the first T-strand, and wherein the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest. In someembodiments, the RB1 and the RB2 are essentially homologous. In someembodiments, the LB1 and the LB2 are essentially homologous. In someembodiments, the RB1 and the RB2 are not essentially homologous. In someembodiments, the LB1 and LB2 are not essentially homologous. In someembodiments, at least one of the RB1 and RB2 comprise an AgrobacteriumTi plasmid right border consensus DNA sequence. In some embodiments, theright border consensus DNA sequence is selected from SEQ ID NO: 21 orSEQ ID NO: 22. In some embodiments, at least one of the RB1 and RB2comprise a sequence selected from SEQ ID NOs: 1-13. In some embodiments,at least one of the RB1 and RB2 comprise a sequence at least 80%identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO:12. Insome embodiments, at least one of the LB1 and LB2 comprise anAgrobacterium Ti plasmid left border consensus DNA sequence. In someembodiments, the left border consensus DNA sequence is selected from SEQID NO: 23 or SEQ ID NO: 24. In some embodiments, at least one of the LB1and LB2 comprise a sequence selected from SEQ ID NOs: 14-20. In someembodiments, at least one of the LB1 and LB2 comprise a sequence atleast 80% identical to SEQ ID NO:19. In some embodiments, the sequenceof interest comprises one or more expression cassettes. In someembodiments, the sequence of interest comprises one or more sequencesselected from: a gene, a portion of a gene, an intergenic sequence, anenhancer, a promoter, an intron, an exon, a sequence encoding atranscription termination sequence, a sequence encoding a chloroplasttargeting peptide, a sequence encoding a mitochondrial targetingpeptide, an insulator sequence, a sequence encoding an anti-sense RNAconstruct, a sequence encoding non-protein-coding RNA (npcRNA), asequence encoding a recombinase, a sequence encoding a recombinaserecognition site, a landing pad, an editing template, an expressioncassette, a stack of two or more expression cassettes encodingtransgenes, a sequence encoding a site-specific enzyme, a sequenceencoding a site-specific enzyme target site, a sequence encoding aselection marker, a sequence encoding a cell factor that functions toincrease DNA repair, a sequence comprising a linker or a spacer, asequence comprising one or more restriction enzyme sites, a sequence fortemplated genome editing, and any combination thereof. In someembodiments, the sequence of interest comprises a sequence encoding asite-specific enzyme target site 5′ to an expression cassette and asequence encoding a site-specific enzyme target site 3′ to an expressioncassette. In some embodiments, the sequence of interest does notcomprise a homology arm DNA sequence. In some embodiments, the sequenceof interest comprises at least one homology arm DNA sequence. In someembodiments, the sequence of interest comprises both a left homology armDNA sequence and a right homology arm DNA sequence. In some embodiments,the sequence of interest comprises i) a first sequence positionedbetween the left homology arm DNA sequence and the right homology DNAarm sequence, and ii) a second sequence that is not positioned betweenthe left homology arm DNA sequence and the right homology arm DNAsequence. In some embodiments, the sequence of interest comprises asequence positioned between the left homology arm DNA sequence and theright homology DNA arm sequence, where the left and right homology armsequences are positioned between two sequences encoding site-specificenzyme target sites. In some embodiments, the sequence of interestcomprises i) a first sequence positioned between the left homology armDNA sequence and the right homology DNA arm sequence where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites, and ii) a second sequencethat is not positioned between sequences encoding site-specific enzymetarget sites. In some embodiments, the sequence of interest comprises i)a first sequence positioned between the left homology arm DNA sequenceand the right homology DNA arm sequence where the left and righthomology arm sequences are positioned between two sequences encodingsite-specific enzyme target sites, and ii) a second sequence encodingthe site-specific enzyme, where the second sequence is not positionedbetween sequences encoding site-specific enzyme target sites. In someembodiments, at least one homology arm DNA sequence comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to a target sequence in the plant genome. In someembodiments, the target sequence in the plant genome is a genicsequence. In some embodiments, the target sequence in the plant genomeis a non-genic sequence. In some embodiments, the sequence of interestflanked by homology arms comprises a sequence that is at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to a native gene of the plant cell. In someembodiments, the sequence of interest comprises a protein-codingsequence. In some embodiments, the sequence of interest comprises anon-protein-coding RNA. In some embodiments, the non-protein-coding RNAis selected from the group consisting of: a microRNA (miRNA), a miRNAprecursor, a small interfering RNA (siRNA), a small RNA (22-26 nt inlength) and precursor encoding same, a heterochromatic siRNA (hc-siRNA),a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpindsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisensesiRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and asingle-guide RNA (sgRNA), or any combination thereof. In someembodiments, the site-specific enzyme is selected from a groupconsisting of an endonuclease, a recombinase, and a transposase. In someembodiments, the endonuclease is selected from a meganuclease, a zincfinger nuclease, a transcription activator-like effector nuclease(TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasYnuclease, and a Cpf1 nuclease. In some embodiments, the Cas9 nuclease isselected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1,Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. Insome embodiments, the Rhizobiales cell further contains a vectorcomprising at least one expression cassette, wherein the expressioncassettes comprise a sequence encoding a protein involved in DNA repair,and wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, the plant cell alreadycomprises a site-specific enzyme. In some embodiments, the plant cell isselected from the group consisting of a corn cell, a soybean cell, acanola cell, a cotton cell, a wheat cell, or a sugarcane cell. In someembodiments, a nucleotide sequence encoding the site-specific enzyme isstably transformed into the plant cell.

Several embodiments relate to a method of transforming a plant cell,comprising contacting the plant cell with two or more Rhizobiales cellscapable of transforming the plant cell, wherein the two or moreRhizobiales cells each contain one of at least two vectors capable offorming two essentially complementary T-strands, wherein each vectorcomprises an essentially identical sequence of interest, and where thefirst vector comprises a RB1 and a first left border DNA sequence (LB1)which are positioned in the first vector to initiate (RB1) and terminate(LB1) synthesis of a first T-strand such that the sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the second vector comprises a RB2 and a second left border DNAsequence (LB2) which are positioned in the second vector to initiate(RB2) and terminate (LB2) synthesis of a second T-strand such that thesequence of interest is in an anti-sense orientation from the 5′ to 3′end of the second T-strand, and wherein the sequence of interest in thetwo T-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other. In some embodiments, the RB1 and the RB2are essentially homologous. In some embodiments, the LB1 and the LB2 areessentially homologous. In some embodiments, the RB1 and the RB2 are notessentially homologous. In some embodiments, the LB1 and LB2 are notessentially homologous. In some embodiments, at least one of the RB1 andRB2 comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Flp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell. In some embodiments, the Rhizobiales cell is selected from anAgrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., anOchrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and aSinorhizobium spp. In some embodiments, the Rhizobiales cell furthercontains a vector comprising at least one expression cassette, whereinthe expression cassettes comprise a sequence encoding a protein involvedin DNA repair, and wherein the protein is selected from the groupcomprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof.

Several embodiments relate to a method of increasing the rate of sitedirected integration of a sequence of interest, comprising contacting aplant cell with at least one vector capable of forming two essentiallycomplementary T-strands, wherein the at least one vector comprises afirst right border DNA sequence (RB1), a second right border DNAsequence (RB2), and at least one sequence of interest, wherein the RB1is positioned in the vector to initiate synthesis of a first T-strandsuch that the sequence of interest is in the sense orientation from the5′ to 3′ end of the first T-strand; and the RB2 is positioned in thevector to initiate synthesis of a second T-strand such that the sequenceof interest is in the anti-sense orientation relative to the sequence ofinterest in the first T-strand, and wherein the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the sequence of interest. In some embodiments, the RB1 andthe RB2 are essentially homologous. In some embodiments, the LB1 and theLB2 are essentially homologous. In some embodiments, the RB1 and the RB2are not essentially homologous. In some embodiments, the LB1 and LB2 arenot essentially homologous. In some embodiments, at least one of the RB1and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Flp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell.

Several embodiments relate to a method of increasing the rate of sitedirected integration of a sequence of interest, comprising contacting aplant cell with at least one vector capable of forming two essentiallycomplementary T-strands, wherein the at least one vector comprises aRB1, a RB2, a sequence of interest, a first left border DNA sequence(LB1), a second left border DNA sequence (LB2) and wherein the vector isconfigured such that the RB1 is paired with the LB1 which are positionedin the vector to initiate (RB1) and terminate (LB1) synthesis of a firstT-strand such that the sequence of interest is in the sense orientationfrom the 5′ to 3′ end of the first T-strand; and the RB2 is paired withthe LB2 which are positioned in the vector to initiate (RB2) andterminate (LB2) synthesis of a second T-strand such that the sequence ofinterest is in an anti-sense orientation relative to the sequence ofinterest in the first T-strand, and wherein the sequence of interest inthe two T-strands resulting from initiation at RB1 and RB2 areessentially complementary in at least a portion of the sequence ofinterest. In some embodiments, the RB1 and the RB2 are essentiallyhomologous. In some embodiments, the LB1 and the LB2 are essentiallyhomologous. In some embodiments, the RB1 and the RB2 are not essentiallyhomologous. In some embodiments, the LB1 and LB2 are not essentiallyhomologous. In some embodiments, at least one of the RB1 and RB2comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Flp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell.

Several embodiments relate to a method of increasing the rate of sitedirected integration of a sequence of interest, comprising contacting aplant cell with at least one vector capable of forming two essentiallycomplementary T-strands, wherein the vector comprises a first sequenceof interest and a second sequence of interest, wherein the firstsequence of interest is essentially identical to the second sequence ofinterest; wherein the vector further comprises a RB1 and a LB1 which arepositioned in the vector to initiate (RB1) and terminate (LB1) synthesisof a first T-strand such that the first sequence of interest is in thesense orientation from the 5′ to 3′ end of the first T-strand; and thevector further comprises a RB2 and aLB2 which are positioned in thevector to initiate (RB2) and terminate (LB2) synthesis of a secondT-strand such that the second sequence of interest is in an anti-senseorientation relative to the first sequence of interest in the firstT-strand, and wherein the two T-strands resulting from initiation at RB1and RB2 are essentially complementary in at least a portion of the firstsequence of interest and the second sequence of interest. In someembodiments, the RB1 and the RB2 are essentially homologous. In someembodiments, the LB1 and the LB2 are essentially homologous. In someembodiments, the RB1 and the RB2 are not essentially homologous. In someembodiments, the LB1 and LB2 are not essentially homologous. In someembodiments, at least one of the RB1 and RB2 comprise an AgrobacteriumTi plasmid right border consensus DNA sequence. In some embodiments, theright border consensus DNA sequence is selected from SEQ ID NO: 21 orSEQ ID NO: 22. In some embodiments, at least one of the RB1 and RB2comprise a sequence selected from SEQ ID NOs: 1-13. In some embodiments,at least one of the RB1 and RB2 comprise a sequence at least 80%identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO:12. Insome embodiments, at least one of the LB1 and LB2 comprise anAgrobacterium Ti plasmid left border consensus DNA sequence. In someembodiments, the left border consensus DNA sequence is selected from SEQID NO: 23 or SEQ ID NO: 24. In some embodiments, at least one of the LB1and LB2 comprise a sequence selected from SEQ ID NOs: 14-20. In someembodiments, at least one of the LB1 and LB2 comprise a sequence atleast 80% identical to SEQ ID NO:19. In some embodiments, the sequenceof interest comprises one or more expression cassettes. In someembodiments, the sequence of interest comprises one or more sequencesselected from: a gene, a portion of a gene, an intergenic sequence, anenhancer, a promoter, an intron, an exon, a sequence encoding atranscription termination sequence, a sequence encoding a chloroplasttargeting peptide, a sequence encoding a mitochondrial targetingpeptide, an insulator sequence, a sequence encoding an anti-sense RNAconstruct, a sequence encoding non-protein-coding RNA (npcRNA), asequence encoding a recombinase, a sequence encoding a recombinaserecognition site, a landing pad, an editing template, an expressioncassette, a stack of two or more expression cassettes encodingtransgenes, a sequence encoding a site-specific enzyme, a sequenceencoding a site-specific enzyme target site, a sequence encoding aselection marker, a sequence encoding a cell factor that functions toincrease DNA repair, a sequence comprising a linker or a spacer, asequence comprising one or more restriction enzyme sites, a sequence fortemplated genome editing, and any combination thereof. In someembodiments, the sequence of interest comprises a sequence encoding asite-specific enzyme target site 5′ to an expression cassette and asequence encoding a site-specific enzyme target site 3′ to an expressioncassette. In some embodiments, the sequence of interest does notcomprise a homology arm DNA sequence. In some embodiments, the sequenceof interest comprises at least one homology arm DNA sequence. In someembodiments, the sequence of interest comprises both a left homology armDNA sequence and a right homology arm DNA sequence. In some embodiments,the sequence of interest comprises i) a first sequence positionedbetween the left homology arm DNA sequence and the right homology DNAarm sequence, and ii) a second sequence that is not positioned betweenthe left homology arm DNA sequence and the right homology arm DNAsequence. In some embodiments, the sequence of interest comprises asequence positioned between the left homology arm DNA sequence and theright homology DNA arm sequence, where the left and right homology armsequences are positioned between two sequences encoding site-specificenzyme target sites. In some embodiments, the sequence of interestcomprises i) a first sequence positioned between the left homology armDNA sequence and the right homology DNA arm sequence where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites, and ii) a second sequencethat is not positioned between sequences encoding site-specific enzymetarget sites. In some embodiments, the sequence of interest comprises i)a first sequence positioned between the left homology arm DNA sequenceand the right homology DNA arm sequence where the left and righthomology arm sequences are positioned between two sequences encodingsite-specific enzyme target sites, and ii) a second sequence encodingthe site-specific enzyme, where the second sequence is not positionedbetween sequences encoding site-specific enzyme target sites. In someembodiments, at least one homology arm DNA sequence comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to a target sequence in the plant genome. In someembodiments, the target sequence in the plant genome is a genicsequence. In some embodiments, the target sequence in the plant genomeis a non-genic sequence. In some embodiments, the sequence of interestflanked by homology arms comprises a sequence that is at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to a native gene of the plant cell. In someembodiments, the sequence of interest comprises a protein-codingsequence. In some embodiments, the sequence of interest comprises anon-protein-coding RNA. In some embodiments, the non-protein-coding RNAis selected from the group consisting of: a microRNA (miRNA), a miRNAprecursor, a small interfering RNA (siRNA), a small RNA (22-26 nt inlength) and precursor encoding same, a heterochromatic siRNA (hc-siRNA),a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpindsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisensesiRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and asingle-guide RNA (sgRNA), or any combination thereof. In someembodiments, the site-specific enzyme is selected from a groupconsisting of an endonuclease, a recombinase, and a transposase. In someembodiments, the endonuclease is selected from a meganuclease, a zincfinger nuclease, a transcription activator-like effector nuclease(TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasYnuclease, and a Cpf1 nuclease. In some embodiments, the Cas9 nuclease isselected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1,Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell.

Several embodiments relate to a method of increasing the rate of sitedirected integration of a sequence of interest, comprising contacting aplant cell with at least one vector capable of forming two essentiallycomplementary T-strands, wherein the Rhizobiales cell comprises at leasta first and a second vector, wherein each vector comprises essentiallyidentical sequences of interest, and wherein the first vector comprisesa RB1 and a LB1 which are positioned in the first vector to initiate(RB1) and terminate (LB1) synthesis of a first T-strand such that thesequence of interest is in the sense orientation from the 5′ to 3′ endof the first T-strand; and the second vector comprises a RB2 and a LB2which are positioned in the second vector to initiate (RB2) andterminate (LB2) synthesis of a second T-strand such that the sequence ofinterest is in an anti-sense orientation relative to the sequence ofinterest in the first T-strand, and wherein the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the sequence of interest. In some embodiments, the RB1 andthe RB2 are essentially homologous. In some embodiments, the LB1 and theLB2 are essentially homologous. In some embodiments, the RB1 and the RB2are not essentially homologous. In some embodiments, the LB1 and LB2 arenot essentially homologous. In some embodiments, at least one of the RB1and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. Insome embodiments, the Rhizobiales cell further contains a vectorcomprising at least one expression cassette, wherein the expressioncassettes comprise a sequence encoding a protein involved in DNA repair,and wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, the plant cell alreadycomprises a site-specific enzyme. In some embodiments, the plant cell isselected from the group consisting of a corn cell, a soybean cell, acanola cell, a cotton cell, a wheat cell, or a sugarcane cell. In someembodiments, a nucleotide sequence encoding the site-specific enzyme isstably transformed into the plant cell.

Several embodiments relate to a method of increasing the rate of sitedirected integration of a sequence of interest, comprising contacting aplant cell with at least one vector capable of forming two essentiallycomplementary T-strands, wherein the at least one vector comprises afirst sequence of interest, a second sequence of interest different fromthe first sequence of interest, at least two RB DNA sequences, and oneor more optional LB DNA sequences, wherein the first RB DNA sequence(RB1) and a first LB DNA sequence (LB1) are positioned in the vector toinitiate (RB1) and terminate (LB1) synthesis of the first T-strand suchthat the first sequence of interest is in the sense orientation from the5′ to 3′ end of the first T-strand; and the vector configuration furthercomprises a second RB DNA sequence (RB2) and a second LB DNA sequence(LB2) which are positioned in the vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the first sequence ofinterest is in an anti-sense orientation from the 5′ to 3′ end of thesecond T-strand, wherein the two T-strands resulting from initiation atRB1 and RB2 are essentially complementary to each other in at least aportion of the first sequence of interest, and wherein the vectorconfiguration further comprises a third RB DNA sequence (RB3) and athird LB DNA sequence (LB3) which are positioned in the vector toinitiate (RB3) and terminate (LB3) synthesis of a third T-strand suchthat the second sequence of interest is in the sense orientation fromthe 5′ to 3′ end of the third T-strand; and the vector configurationfurther comprises a fourth RB DNA sequence (RB4) and a fourth LB DNAsequence (LB4) which are positioned in the vector to initiate (RB4) andterminate (LB4) synthesis of a fourth T-strand such that the secondsequence of interest is in an anti-sense orientation from the 5′ to 3′end of the fourth T-strand, and the two T-strands resulting frominitiation at RB3 and RB4 are essentially complementary to each other inat least a portion of the second sequence of interest. In someembodiments, the RB1 and the RB2 are essentially homologous. In someembodiments, the LB1 and the LB2 are essentially homologous. In someembodiments, the RB1 and the RB2 are not essentially homologous. In someembodiments, the LB1 and LB2 are not essentially homologous. In someembodiments, at least one of the RB1 and RB2 comprise an AgrobacteriumTi plasmid right border consensus DNA sequence. In some embodiments, theright border consensus DNA sequence is selected from SEQ ID NO: 21 orSEQ ID NO: 22. In some embodiments, at least one of the RB1 and RB2comprise a sequence selected from SEQ ID NOs: 1-13. In some embodiments,at least one of the RB1 and RB2 comprise a sequence at least 80%identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO:12. Insome embodiments, at least one of the LB1 and LB2 comprise anAgrobacterium Ti plasmid left border consensus DNA sequence. In someembodiments, the left border consensus DNA sequence is selected from SEQID NO: 23 or SEQ ID NO: 24. In some embodiments, at least one of the LB1and LB2 comprise a sequence selected from SEQ ID NOs: 14-20. In someembodiments, at least one of the LB1 and LB2 comprise a sequence atleast 80% identical to SEQ ID NO:19. In some embodiments, the sequenceof interest comprises one or more expression cassettes. In someembodiments, the sequence of interest comprises one or more sequencesselected from: a gene, a portion of a gene, an intergenic sequence, anenhancer, a promoter, an intron, an exon, a sequence encoding atranscription termination sequence, a sequence encoding a chloroplasttargeting peptide, a sequence encoding a mitochondrial targetingpeptide, an insulator sequence, a sequence encoding an anti-sense RNAconstruct, a sequence encoding non-protein-coding RNA (npcRNA), asequence encoding a recombinase, a sequence encoding a recombinaserecognition site, a landing pad, an editing template, an expressioncassette, a stack of two or more expression cassettes encodingtransgenes, a sequence encoding a site-specific enzyme, a sequenceencoding a site-specific enzyme target site, a sequence encoding aselection marker, a sequence encoding a cell factor that functions toincrease DNA repair, a sequence comprising a linker or a spacer, asequence comprising one or more restriction enzyme sites, a sequence fortemplated genome editing, and any combination thereof. In someembodiments, the sequence of interest comprises a sequence encoding asite-specific enzyme target site 5′ to an expression cassette and asequence encoding a site-specific enzyme target site 3′ to an expressioncassette. In some embodiments, the sequence of interest does notcomprise a homology arm DNA sequence. In some embodiments, the sequenceof interest comprises at least one homology arm DNA sequence. In someembodiments, the sequence of interest comprises both a left homology armDNA sequence and a right homology arm DNA sequence. In some embodiments,the sequence of interest comprises i) a first sequence positionedbetween the left homology arm DNA sequence and the right homology DNAarm sequence, and ii) a second sequence that is not positioned betweenthe left homology arm DNA sequence and the right homology arm DNAsequence. In some embodiments, the sequence of interest comprises asequence positioned between the left homology arm DNA sequence and theright homology DNA arm sequence, where the left and right homology armsequences are positioned between two sequences encoding site-specificenzyme target sites. In some embodiments, the sequence of interestcomprises i) a first sequence positioned between the left homology armDNA sequence and the right homology DNA arm sequence where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites, and ii) a second sequencethat is not positioned between sequences encoding site-specific enzymetarget sites. In some embodiments, the sequence of interest comprises i)a first sequence positioned between the left homology arm DNA sequenceand the right homology DNA arm sequence where the left and righthomology arm sequences are positioned between two sequences encodingsite-specific enzyme target sites, and ii) a second sequence encodingthe site-specific enzyme, where the second sequence is not positionedbetween sequences encoding site-specific enzyme target sites. In someembodiments, at least one homology arm DNA sequence comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to a target sequence in the plant genome. In someembodiments, the target sequence in the plant genome is a genicsequence. In some embodiments, the target sequence in the plant genomeis a non-genic sequence. In some embodiments, the sequence of interestflanked by homology arms comprises a sequence that is at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to a native gene of the plant cell. In someembodiments, the sequence of interest comprises a protein-codingsequence. In some embodiments, the sequence of interest comprises anon-protein-coding RNA. In some embodiments, the non-protein-coding RNAis selected from the group consisting of: a microRNA (miRNA), a miRNAprecursor, a small interfering RNA (siRNA), a small RNA (22-26 nt inlength) and precursor encoding same, a heterochromatic siRNA (hc-siRNA),a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpindsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisensesiRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and asingle-guide RNA (sgRNA), or any combination thereof. In someembodiments, the site-specific enzyme is selected from a groupconsisting of an endonuclease, a recombinase, and a transposase. In someembodiments, the endonuclease is selected from a meganuclease, a zincfinger nuclease, a transcription activator-like effector nuclease(TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasYnuclease, and a Cpf1 nuclease. In some embodiments, the Cas9 nuclease isselected from the group comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1,Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell.

Several embodiments relate to a method of transforming a plant cell,comprising contacting the plant cell with a Rhizobiales cell capable oftransforming the plant cell, wherein the Rhizobiales cell comprises atleast one vector comprising a right border DNA sequence (RB) and a leftborder DNA sequence (LB) and wherein the vector comprises between the RBand LB: (i) a first sequence of interest in a sense orientation relativeto the RB, (ii) a spacer, and (iii) a second sequence of interest in ananti-sense orientation relative to the RB, wherein the first sequence ofinterest and second sequence of interest are essentially complementaryand after synthesis of the T-strand anneal to form a double-strandedDNA. In some embodiments, the first sequence of interest furthercomprises a first left homology arm DNA sequence and a first righthomology arm DNA sequence, and the second sequence of interest furthercomprises a second left homology arm DNA sequence and a second righthomology arm DNA sequence. In some embodiments, the Rhizobiales cell isselected from an Agrobacterium spp., a Bradyrhizobium spp., aMesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., aRhizobium spp., and a Sinorhizobium spp. In some embodiments, theRhizobiales cell further contains a vector comprising at least oneexpression cassette, wherein the expression cassettes comprise asequence encoding a protein involved in DNA repair, and wherein theprotein is selected from the group comprising a vir gene from the Tiplasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or anycombination thereof. In some embodiments, the RB1 and the RB2 areessentially homologous. In some embodiments, the LB1 and the LB2 areessentially homologous. In some embodiments, the RB1 and the RB2 are notessentially homologous. In some embodiments, the LB1 and LB2 are notessentially homologous. In some embodiments, at least one of the RB1 andRB2 comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. Insome embodiments, the Rhizobiales cell further contains a vectorcomprising at least one expression cassette, wherein the expressioncassettes comprise a sequence encoding a protein involved in DNA repair,and wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, the plant cell alreadycomprises a site-specific enzyme. In some embodiments, the plant cell isselected from the group consisting of a corn cell, a soybean cell, acanola cell, a cotton cell, a wheat cell, or a sugarcane cell. In someembodiments, a nucleotide sequence encoding the site-specific enzyme isstably transformed into the plant cell.

Several embodiments relate to a method of increasing the rate of sitedirected integration of a double-stranded DNA into a genome of a plantcell, comprising contacting the plant cell with at least one vectorcomprising a right border DNA sequence (RB) and a left border DNAsequence (LB), wherein the vector comprises between the RB and LB: (i) afirst sequence of interest in a sense orientation relative to the RB,(ii) a spacer, and (iii) a second sequence of interest in an anti-senseorientation relative to the RB, wherein the first sequence of interestand second sequence of interest are essentially complementary and aftersynthesis of the T-strand anneal to form a double-stranded DNA. In someembodiments, the first sequence of interest further comprises a firstleft homology arm DNA sequence and a first right homology arm DNAsequence, and the second sequence of interest further comprises a secondleft homology arm DNA sequence and a second right homology arm DNAsequence. In some embodiments, the RB1 and the RB2 are essentiallyhomologous. In some embodiments, the LB1 and the LB2 are essentiallyhomologous. In some embodiments, the RB1 and the RB2 are not essentiallyhomologous. In some embodiments, the LB1 and LB2 are not essentiallyhomologous. In some embodiments, at least one of the RB1 and RB2comprise an Agrobacterium Ti plasmid right border consensus DNAsequence. In some embodiments, the right border consensus DNA sequenceis selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, atleast one of the RB1 and RB2 comprise a sequence selected from SEQ IDNOs: 1-13. In some embodiments, at least one of the RB1 and RB2 comprisea sequence at least 80% identical to a sequence selected from SEQ IDNOs:4 and SEQ ID NO:12. In some embodiments, at least one of the LB1 andLB2 comprise an Agrobacterium Ti plasmid left border consensus DNAsequence. In some embodiments, the left border consensus DNA sequence isselected from SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, atleast one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20. In some embodiments, at least one of the LB1 and LB2comprise a sequence at least 80% identical to SEQ ID NO:19. In someembodiments, the sequence of interest comprises one or more expressioncassettes. In some embodiments, the sequence of interest comprises oneor more sequences selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof. In some embodiments, the sequence of interest comprises asequence encoding a site-specific enzyme target site 5′ to an expressioncassette and a sequence encoding a site-specific enzyme target site 3′to an expression cassette. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest comprises at least one homology arm DNA sequence.In some embodiments, the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence. In someembodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology DNA arm sequence, and ii) a second sequence that is notpositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence. In some embodiments, the sequence of interestcomprises a sequence positioned between the left homology arm DNAsequence and the right homology DNA arm sequence, where the left andright homology arm sequences are positioned between two sequencesencoding site-specific enzyme target sites. In some embodiments, thesequence of interest comprises i) a first sequence positioned betweenthe left homology arm DNA sequence and the right homology DNA armsequence where the left and right homology arm sequences are positionedbetween two sequences encoding site-specific enzyme target sites, andii) a second sequence that is not positioned between sequences encodingsite-specific enzyme target sites. In some embodiments, the sequence ofinterest comprises i) a first sequence positioned between the lefthomology arm DNA sequence and the right homology DNA arm sequence wherethe left and right homology arm sequences are positioned between twosequences encoding site-specific enzyme target sites, and ii) a secondsequence encoding the site-specific enzyme, where the second sequence isnot positioned between sequences encoding site-specific enzyme targetsites. In some embodiments, at least one homology arm DNA sequencecomprises a sequence that is at least 80%, at least 85%, at least 90%,at least 95%, or at least 99% identical to a target sequence in theplant genome. In some embodiments, the target sequence in the plantgenome is a genic sequence. In some embodiments, the target sequence inthe plant genome is a non-genic sequence. In some embodiments, thesequence of interest flanked by homology arms comprises a sequence thatis at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a native gene of the plantcell. In some embodiments, the sequence of interest comprises aprotein-coding sequence. In some embodiments, the sequence of interestcomprises a non-protein-coding RNA. In some embodiments, thenon-protein-coding RNA is selected from the group consisting of: amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the site-specific enzyme isselected from a group consisting of an endonuclease, a recombinase, anda transposase. In some embodiments, the endonuclease is selected from ameganuclease, a zinc finger nuclease, a transcription activator-likeeffector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasXnuclease, a CasY nuclease, and a Cpf1 nuclease. In some embodiments, theCas9 nuclease is selected from the group comprising Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4nuclease. In some embodiments, the recombinase is a tyrosine recombinaseattached to a DNA recognition motif, or a serine recombinase attached toa DNA recognition motif. In some embodiments, the tyrosine recombinaseattached to a DNA recognition motif is selected from the groupconsisting of a Cre recombinase, a Pp recombinase, and a Tnp1recombinase. In some embodiments, the serine recombinase attached to aDNA recognition motif is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In some embodiments,the transposase is a DNA transposase attached to a DNA binding domain.In some embodiments, the sequence of interest further comprises at leastone site-specific enzyme target site. In some embodiments, the at leastone site-specific enzyme target site is selected from a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site. In some embodiments, the sequence ofinterest comprises a sequence encoding a protein involved in DNA repair,wherein the protein is selected from the group comprising a vir genefrom the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, orany combination thereof. In some embodiments, at least part of thesequence of interest is integrated into the plant genome via homologousrecombination, wherein the integration of at least part of the sequenceof interest results in a point mutation, an insertion, a deletion, aninversion, increased transcription of an endogenous locus, decreasedtranscription of an endogenous locus, altered protein activity, alteredRNAi products, altered RNAi target sites, altered RNAi pathway activity,increased transcription of the sequence of interest, decreasedtranscription of the integrated sequence of interest, or a combinationthereof. In some embodiments, at least part of the sequence of interestis integrated into the plant genome via non-homologous end joining,wherein the integration of at least part of the sequence of interestresults in a point mutation, an insertion, a deletion, an inversion,increased transcription of an endogenous locus, decreased transcriptionof an endogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or a combination thereof. In someembodiments, the plant cell already comprises a site-specific enzyme. Insome embodiments, the plant cell is selected from the group consistingof a corn cell, a soybean cell, a canola cell, a cotton cell, a wheatcell, or a sugarcane cell. In some embodiments, a nucleotide sequenceencoding the site-specific enzyme is stably transformed into the plantcell.

Several embodiments relate to an Agrobacterium cell comprising at leastone vector that is capable of forming two essentially complementaryT-strands. In some embodiments, the Agrobacterium cell comprises atleast one vector comprises a first right border DNA sequence (RB1), asecond right border DNA sequence (RB2), and at least one sequence ofinterest, and wherein the RB1 is positioned in the vector to initiatesynthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andthe RB2 is positioned in the vector to initiate synthesis of a secondT-strand such that the sequence of interest is in the anti-senseorientation relative to the sequence of interest in the first T-strand,and wherein the two T-strands resulting from initiation at RB1 and RB2are essentially complementary in at least a portion of the sequence ofinterest. In some embodiments, the Agrobacterium cell comprises at leastone vector comprises a first right border DNA sequence (RB1), a secondright border DNA sequence (RB2), a sequence of interest, a first leftborder DNA sequence (LB1) and a second left border DNA sequence (LB2),wherein the vector is configured such that the RB1 is paired with theLB1 which are positioned in the vector to initiate (RB1) and terminate(LB1) synthesis of a first T-strand such that the sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the RB2 is paired with the LB2 which are positioned in the vector toinitiate (RB2) and terminate (LB2) synthesis of a second T-strand suchthat the sequence of interest is in an anti-sense orientation relativeto the sequence of interest in the first T-strand, and wherein the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest. In someembodiments, the Agrobacterium cell comprises at least one vectorcomprises a first sequence of interest and a second sequence ofinterest, where the first sequence of interest is essentially identicalto the second sequence of interest; wherein the vector further comprisesa first right border DNA sequence (RB1) and a first left border DNAsequence (LB1) which are positioned in the vector to initiate (RB1) andterminate (LB1) synthesis of a first T-strand such that the firstsequence of interest is in the sense orientation from the 5′ to 3′ endof the first T-strand; and the vector further comprises a second rightborder DNA sequence (RB2) and a second left border DNA sequence (LB2)which are positioned in the vector to initiate (RB2) and terminate (LB2)synthesis of a second T-strand such that the second sequence of interestis in an anti-sense orientation relative to the first sequence ofinterest in the first T-strand, and wherein the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the first sequence of interest and the second sequence ofinterest. In some embodiments, the Agrobacterium cell comprises at leasta first vector and a second vector wherein each vector comprisesessentially identical sequences of interest, and wherein the firstvector comprises a first right border DNA sequence (RB1) and a firstleft border DNA sequence (LB1) which are positioned in the first vectorto initiate (RB1) and terminate (LB1) synthesis of a first T-strand suchthat the sequence of interest is in the sense orientation from the 5′ to3′ end of the first T-strand; and wherein the second vector comprises asecond right border DNA sequence (RB2) and a second left border DNAsequence (LB2) which are positioned in the second vector to initiate(RB2) and terminate (LB2) synthesis of a second T-strand such that thesequence of interest is in an anti-sense orientation relative to thesequence of interest in the first T-strand, and wherein the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest. In someembodiments, the Agrobacterium cell comprises a RB1 and RB2 that areessentially homologous. In some embodiments, the Agrobacterium cellcomprises a LB1 and LB2 that are essentially homologous. In someembodiments, the Agrobacterium cell comprises RB1 and RB2 sequences thatare not essentially homologous. In some embodiments, the Agrobacteriumcell of LB1 and LB2 sequences that are not essentially homologous. Insome embodiments, at least one of the RB1 and RB2 comprise anAgrobacterium Ti plasmid right border consensus DNA sequence. In someembodiments, the right border consensus DNA sequence is selected fromSEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of theRB1 and RB2 comprise a sequence selected from SEQ ID NOs: 1-13. In someembodiments, at least one of the RB1 and RB2 comprise a sequence atleast 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ IDNO:12. In some embodiments, at least one of the LB1 and LB2 comprise anAgrobacterium Ti plasmid left border consensus DNA sequence. In someembodiments, the left border consensus DNA sequence is selected from SEQID NO: 23 or SEQ ID NO: 24 In some embodiments, at least one of the LB1and LB2 comprise a sequence selected from SEQ ID NOs: 14-20. In someembodiments, at least one of the LB1 and LB2 comprise a sequence atleast 80% identical to SEQ ID NO: 19. In some embodiments, the sequenceof interest comprises one or more expression cassettes. In someembodiments, the sequence of interest comprises at least one sequenceselected from: a gene, a portion of a gene, an intergenic sequence, anenhancer, a promoter, an intron, an exon, a sequence encoding atranscription termination sequence, a sequence encoding a chloroplasttargeting peptide, a sequence encoding a mitochondrial targetingpeptide, an insulator sequence, a sequence encoding an anti-sense RNAconstruct, a sequence encoding non-protein-coding RNA (npcRNA), asequence encoding a recombinase, a sequence encoding a recombinaserecognition site, a landing pad, an editing template, an expressioncassette, a stack of two or more expression cassettes encodingtransgenes, a sequence encoding a site-specific enzyme, a sequenceencoding a site-specific enzyme target site, a sequence encoding aselection marker, gene expression cassette comprising a sequenceencoding a cell factor that functions to increase DNA repair, a sequencecomprising a linker or a spacer, a sequence comprising one or morerestriction enzyme sites, a sequence for templated genome editing, andany combination thereof. In some embodiments, the sequence of interestdoes not comprise a homology arm DNA sequence. In some embodiments, thesequence of interest further comprises at least one homology arm DNAsequence. In some embodiments, the sequence of interest comprises both aleft homology arm DNA sequence and a right homology arm DNA sequence. Insome embodiments, the sequence of interest comprises i) a first sequencepositioned between the left homology arm DNA sequence and the righthomology arm DNA sequence, and ii) a second sequence that is notpositioned between the region comprising the left homology arm DNAsequence and the right homology arm DNA sequence In some embodiments,the at least one homology arm DNA sequence comprises a sequence that isat least 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to a target sequence in the plant genome. In some embodiments,the sequence of interest comprises a protein-coding sequence. In someembodiments, the sequence of interest comprises a non-protein-coding RNAIn some embodiments, the non-protein-coding RNA is selected from amicroRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), asmall RNA (22-26 nt in length) and precursor encoding same, aheterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), ahairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA(ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracerRNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or anycombination thereof. In some embodiments, the expression cassettecomprises a nucleic acid sequence at least 80% identical to a nativeplant gene. In some embodiments, the expression cassette comprises anucleic acid sequence that is not homologous to a native plant sequence.In some embodiments, at least one expression cassette comprises at asequence selected from: an insecticidal resistance gene, herbicidetolerance gene, nitrogen use efficiency gene, a water use efficiencygene, a nutritional quality gene, a DNA binding gene, a selectablemarker gene, an RNAi construct, a site specific nuclease gene, a guideRNA, and any combination thereof. In some embodiments, the site-specificenzyme is selected from an endonuclease, a recombinase, a transposase,and any combination thereof. In some embodiments, the endonuclease isselected from the group consisting of: a meganuclease, a zinc fingernuclease, a transcription activator-like effector nuclease (TALEN), anArgonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpf1nuclease. In some embodiments, the Cas9 nuclease is selected from thegroup comprising Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8,Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1,Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3,Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX,Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4 nuclease. In someembodiments, the recombinase is a tyrosine recombinase attached to a DNArecognition motif, or a serine recombinase attached to a DNA recognitionmotif. In some embodiments, the tyrosine recombinase attached to a DNArecognition motif is selected from the group consisting of a Crerecombinase, a Flp recombinase, and a Tnp1 recombinase. In someembodiments, the serine recombinase attached to a DNA recognition motifis selected from the group consisting of a PhiC31 integrase, an R4integrase, and a TP-901 integrase. In some embodiments, the transposaseis a DNA transposase attached to a DNA binding domain. In someembodiments, the sequence of interest comprises at least onesite-specific enzyme target site. In some embodiments, at least onesite-specific enzyme target site is selected from the group consistingof: a Cre/lox recombination site, a Flp/FRT recombination site, aendonuclease recognition site, and a TALEN site. In some embodiments,the sequence of interest comprises a sequence encoding at least oneprotein involved in DNA repair, wherein the protein is selected from thegroup comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof.

Several embodiments relate to an Agrobacterium cell comprising at leastone vector comprising a right border DNA sequence (RB) and a left borderDNA sequence (LB) and wherein the vector comprises between the RB andLB: (i) a first sequence of interest in a sense orientation relative tothe RB, (ii) a spacer, and (iii) a second sequence of interest in ananti-sense orientation relative to the RB, wherein the first sequence ofinterest and second sequence of interest are essentially complementary,and after synthesis of the T-strand anneal to form a double-strandedDNA. Ins some embodiments, the Agrobacterium cell comprises a firstsequence of interest comprising a left homology arm DNA sequence and aright homology arm DNA sequence and a second sequence of interestcomprising a left homology arm sequence DNA sequence and a righthomology arm DNA sequence. In some embodiments, the RB comprises anAgrobacterium Ti plasmid right border consensus DNA sequence. In someembodiments, the right border consensus DNA sequence is selected fromSEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, the RB comprises asequence selected from SEQ ID NOs: 1-13. In some embodiments, the RBcomprises a sequence at least 80% identical to a sequence selected fromSEQ ID NOs:4 and SEQ ID NO:12. In some embodiments, the LB comprises anAgrobacterium Ti plasmid left border consensus DNA sequence. In someembodiments, the left border consensus DNA sequence is selected from SEQID NO: 23 or SEQ ID NO: 24. In some embodiments, the LB comprises asequence selected from SEQ ID NOs: 14-20. In some embodiments, the LBcomprises a sequence at least 80% identical to SEQ ID NO: 19. In someembodiments, a sequence of interest comprises one or more expressioncassettes. In some embodiments, a sequence of interest comprises atleast one sequence selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, asequence encoding a transcription termination sequence, a sequenceencoding a chloroplast targeting peptide, a sequence encoding amitochondrial targeting peptide, an insulator sequence, a sequenceencoding an anti-sense RNA construct, a sequence encodingnon-protein-coding RNA (npcRNA), a sequence encoding a recombinase, asequence encoding a recombinase recognition site, a landing pad, anediting template, an expression cassette, a stack of two or moreexpression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, gene expression cassettecomprising a sequence encoding a cell factor that functions to increaseDNA repair, a sequence comprising a linker or a spacer, a sequencecomprising one or more restriction enzyme sites, a sequence fortemplated genome editing, and any combination thereof. In someembodiments, a sequence of interest does not comprise a homology arm DNAsequence. In some embodiments, a sequence of interest further comprisesat least one homology arm DNA sequence.

Several embodiments relate to a method of transforming a plant genome,comprising contacting at least one plant cell on a co-culture medium forat least 24-48 hours, for at least 24-30 hours, for at least 30-36hours, for at least 36-42 hours, for at least 42-48 hours, for at least48-54 hours, for at least 54-60 hours, for at least 60-66 hours, for atleast 66-72 hours, for at least 72-78 hours, for at least 78-84 hours,for at least 84-90 hours, for at least 90-96 hours, for at least 96-102hours, for at least 102-108 hours, for at least 108-114 hours, for atleast 114-120 hours, for at least 120-126 hours, or for at least 126-132hours, with at least one Rhizobiales cell capable of transforming theplant cell, In some embodiments, the Rhizobiales cell comprises at leastone vector capable of forming two essentially complementary T-strands.In some embodiments, the plant cell is a corn immature embryo cell, acorn mature embryo cell, a corn seed cell, a soybean immature embryocell, a soybean mature embryo cell, a soybean seed cell, a canolaimmature embryo cell, a canola mature embryo cell, a canola seed cell, acotton immature embryo cell, a cotton mature embryo cell, a cotton seedcell, a wheat immature embryo cell, a wheat mature embryo cell, a wheatseed cell, a sugarcane immature embryo cell, a sugarcane mature embryocell, or a sugarcane seed cell. In some embodiments, the Rhizobialescell comprises at least one vector comprises a first right border (RB1)DNA sequence, a second right border DNA sequence (RB2), and at least onesequence of interest, and wherein the RB1 is positioned in the vector toinitiate synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the RB2 is positioned in the vector to initiate synthesisof a second T-strand such that the sequence of interest is in theanti-sense orientation relative to the sequence of interest in the firstT-strand, and wherein the sequence of interest in the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryin at least a portion of the sequence of interest. In some embodiments,the Rhizobiales cell comprises at least one vector disclosed hereincomprises a RB1, a RB2, and a sequence of interest, and furthercomprises a first left border DNA sequence (LB1) and a second leftborder DNA sequence (LB2), and wherein the vector is configured suchthat the RB1 is paired with the LB1 which are positioned in the vectorto initiate (RB1) and terminate (LB1) synthesis of a first T-strand suchthat the sequence of interest is in the sense orientation from the 5′ to3′ end of the first T-strand; and the RB2 is paired with the LB2 whichare positioned in the vector to initiate (RB2) and terminate (LB2)synthesis of a second T-strand such that the sequence of interest is inan anti-sense orientation relative to the sequence of interest in thefirst T-strand, and wherein the sequence of interest in the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest. In someembodiments, the Rhizobiales cell comprises a vector comprising a firstsequence of interest and a second sequence of interest, where the firstsequence of interest is essentially identical to the second sequence ofinterest; and the vector configuration further comprises a RB1 with aLB1which are positioned in the vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the first sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the vector further comprises a RB2 and a LB2 which are positioned inthe vector to initiate (RB2) and terminate (LB2) synthesis of a secondT-strand such that the second sequence of interest is in an anti-senseorientation relative to the first sequence of interest in the firstT-strand, and wherein the sequence of interest in the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryin at least a portion of the first sequence of interest and the secondsequence of interest. In some embodiments, the Rhizobiales cellcomprises at least a first vector and a second vector, wherein eachvector comprises essentially identical sequences of interest, andwherein the first vector configuration comprises a RB1 and a LB1 whichare positioned in the first vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andwherein the second vector configuration comprises a RB2 and a LB2 whichare positioned in the second vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the sequence of interestis in an anti-sense orientation relative to the sequence of interest inthe first T-strand, and wherein the sequence of interest in the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest. In someembodiments, the Rhizobiales cell comprises at least one vectorcomprises a first sequence of interest, a second sequence of interestdifferent from the first sequence of interest, at least two RB DNAsequences, and one or more optional LB DNA sequences, wherein the firstRB DNA sequence (RB1) and a first LB DNA sequence (LB1) are positionedin the vector to initiate (RB1) and terminate (LB1) synthesis of thefirst T-strand such that the first sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the vectorconfiguration further comprises a second RB DNA sequence (RB2) and asecond LB DNA sequence (LB2) which are positioned in the vector toinitiate (RB2) and terminate (LB2) synthesis of a second T-strand suchthat the first sequence of interest is in an anti-sense orientation fromthe 5′ to 3′ end of the second T-strand, wherein the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryto each other in at least a portion of the first sequence of interest,and wherein the vector configuration further comprises a third RB DNAsequence (RB3) and a third LB DNA sequence (LB3) which are positioned inthe vector to initiate (RB3) and terminate (LB3) synthesis of a thirdT-strand such that the second sequence of interest is in the senseorientation from the 5′ to 3′ end of the third T-strand; and the vectorconfiguration further comprises a fourth RB DNA sequence (RB4) and afourth LB DNA sequence (LB4) which are positioned in the vector toinitiate (RB4) and terminate (LB4) synthesis of a fourth T-strand suchthat the second sequence of interest is in an anti-sense orientationfrom the 5′ to 3′ end of the fourth T-strand, and the two T-strandsresulting from initiation at RB3 and RB4 are essentially complementaryto each other in at least a portion of the second sequence of interest.In some embodiments, the method comprises contacting the plant cell witha first Rhizobiales cell and a second Rhizobiales cell, wherein eachRhizobiales cell contains at least one of two vectors, wherein eachvector comprises an essentially identical sequence of interest, andwhere the first vector comprises a RB1, and wherein the RB1 ispositioned in the vector to initiate synthesis of a first T-strand suchthat the sequence of interest is in the sense orientation from the 5′ to3′ end of the first T-strand; and the second vector comprises a RB2which is positioned in the vector to initiate synthesis of a secondT-strand such that the sequence of interest is in the anti-senseorientation relative to the sequence of interest in the first T-strand,and wherein the two T-strands resulting from initiation at RB1 and RB2are essentially complementary in at least a portion of the sequence ofinterest. In some embodiments, the method comprises contacting the plantcell with a first Rhizobiales cell and a second Rhizobiales cell,wherein each Rhizobiales cell contains at least one of two vectors,wherein each vector comprises an essentially identical sequence ofinterest, and where the first vector comprises a RB1 and a LB1 which arepositioned in the first vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andthe second vector comprises a RB2 and a LB2 which are positioned in thesecond vector to initiate (RB2) and terminate (LB2) synthesis of asecond T-strand such that the sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand, and whereinthe sequence of interest in the two T-strands resulting from initiationat RB1 and RB2 are essentially complementary to each other. In someembodiments, the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. Insome embodiments, the Agrobacterium spp. cell is selected from the groupconsisting of an Agrobacterium tumefaciens cell and an Agrobacteriumrhizogenes cell. In some embodiments, the Rhizobiales cell furthercontains a vector comprising at least one expression cassette, whereinthe expression cassettes comprise a sequence encoding a protein involvedin DNA repair, and wherein the protein is selected from the groupcomprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof. In some embodimentsthe method results in at least a fragment of the sequence of interestbeing integrated into the plant genome by homologous recombination. Insome embodiments, the method results in at least a fragment of thesequence of interest being integrated into the plant genome bynon-homologous end joining. In some embodiments, the method furthercomprises detecting the integration of at least a fragment of thesequence of interest of the vector in the at least one plant cell. Insome embodiments, the method further comprises selecting the plant cellbased on the presence of the at least a fragment of the sequence ofinterest integrated into the plant genome. In some embodiments, themethod further comprises regenerating a transgenic plant from theselected plant cell.

Several embodiments relate to a method of transforming a plant genome,comprising contacting at least one plant cell on a co-culture medium forat least 24-48 hours, for at least 24-30 hours, for at least 30-36hours, for at least 36-42 hours, for at least 42-48 hours, for at least48-54 hours, for at least 54-60 hours, for at least 60-66 hours, for atleast 66-72 hours, for at least 72-78 hours, for at least 78-84 hours,for at least 84-90 hours, for at least 90-96 hours, for at least 96-102hours, for at least 102-108 hours, for at least 108-114 hours, for atleast 114-120 hours, for at least 120-126 hours, or for at least 126-132hours, with at least one Rhizobiales cell capable of transforming theplant cell, wherein the Rhizobiales cell comprises at least one vectorcomprising a right border (RB) DNA sequence and a left border (LB) DNAsequence and where the vector further comprises between the RB and LBDNA sequences: (i) a first sequence of interest in a sense orientationrelative to the RB DNA sequence, and (ii) a spacer, and (iii) a secondsequence of interest in an anti-sense orientation relative to the RB DNAsequence, wherein the first sequence of interest and second sequence ofinterest are essentially complementary, and after synthesis of theT-strand anneal to form a double-stranded DNA. In some embodiments, thefirst sequence of interest further comprises a left homology arm DNAsequence and a right homology arm DNA sequence in and the secondsequence of interest further comprises a left homology arm DNA sequenceand a right homology arm DNA sequence. In some embodiments, theRhizobiales cell is selected from an Agrobacterium spp., aBradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., aPhyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. In someembodiments, the Agrobacterium spp. cell is selected from the groupconsisting of an Agrobacterium tumefaciens cell and an Agrobacteriumrhizogenes cell. In some embodiments, the Rhizobiales cell furthercontains a vector comprising at least one expression cassette, whereinthe expression cassettes comprise a sequence encoding a protein involvedin DNA repair, and wherein the protein is selected from the groupcomprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof. In some embodimentsthe method results in at least a fragment of the sequence of interestbeing integrated into the plant genome by homologous recombination. Insome embodiments, the method results in at least a fragment of thesequence of interest being integrated into the plant genome bynon-homologous end joining. In some embodiments, the method furthercomprises detecting the integration of at least a fragment of thesequence of interest of the vector in the at least one plant cell. Insome embodiments, the method further comprises selecting the plant cellbased on the presence of the at least a fragment of the sequence ofinterest integrated into the plant genome. In some embodiments, themethod further comprises regenerating a transgenic plant from theselected plant cell.

BRIEF DESCRIPTION OF FIGURES

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the aspects of this disclosure andtogether with the description, serve to explain embodiments described inthe disclosure. In the drawings:

FIGS. 1A-1B illustrate two control vector configurations in which asequence of interest is flanked by a right border (RB) DNA sequence onthe 5′ end, and a left border (LB) DNA sequence on the 3′ end (FIG. 1A);or alternatively, one RB DNA sequence on the 5′ end (FIG. 1B).

FIG. 1C illustrates a vector configuration comprising two RB DNAsequences, where the first RB DNA sequence (RB1) is positioned in thevector to initiate synthesis of a first T-strand such that the sequenceof interest is in the sense orientation from the 5′ to 3′ end of thefirst T-strand; and the second RB DNA sequence (RB2) is positioned inthe vector to initiate synthesis of a second T-strand such that thesequence of interest is in the anti-sense orientation from the 5′ to 3′end of the second T-strand. The two T-strands resulting from initiationat RB1 and RB2 are essentially complementary to each other in at least aportion of the sequence of interest.

FIG. 1D illustrates a vector configuration comprising optional LB DNAsequences. In the illustrated vector configuration, the first RB DNAsequence (RB1) is paired with a first LB DNA sequence (LB1) which arepositioned in the vector to initiate (RB1) and terminate (LB1) synthesisof a first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the secondRB DNA sequence (RB2) is paired with a second left border DNA sequence(LB2) which are positioned in the vector to initiate synthesis of asecond T-strand such that the sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand. The twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other in at least a portion of the sequence ofinterest.

FIG. 2A illustrates a vector configuration comprising a first sequenceof interest and a second sequence of interest, where the first sequenceof interest is essentially identical to the second sequence of interest;and the vector configuration further comprises a first RB DNA sequence(RB1) with an optional first LB DNA sequence (LB1) which are positionedin the vector to initiate (RB1) and optionally terminate (LB1) synthesisof a first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the vectorconfiguration further comprises a second RB DNA sequence (RB2) and anoptional second LB DNA sequence (LB2) which are positioned in the vectorto initiate (RB2) and optionally terminate (LB2) synthesis of a secondT-strand such that the sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand. The sequenceof interest in the two T-strands resulting from initiation at RB1 andRB2 are essentially complementary to each other.

FIG. 2B illustrates a vector configuration employed in co-transformationin which two essentially identical sequences of interest are located onseparate vectors, hosted by one or more bacterium cells, and where thefirst vector configuration comprises a first RB DNA sequence (RB1) andan optional first LB DNA sequence (LB1) which are positioned in thefirst vector to initiate (RB1) and optionally terminate (LB1) synthesisof a first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the secondvector configuration comprises a second RB DNA sequence (RB2) and anoptional second LB DNA sequence (LB2) which are positioned in the secondvector to initiate (RB2) and optionally terminate (LB2) synthesis of asecond T-strand such that the sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand. The sequenceof interest in the two T-strands resulting from initiation at RB1 andRB2 are essentially complementary to each other.

FIG. 2C illustrates a vector comprising a first sequence of interest, asecond sequence of interest different from the first sequence ofinterest, two or more RB DNA sequences, and one or more optional LB DNAsequences. The illustrated construct is one non-limiting example wherethe vector configuration comprises a first RB DNA sequence (RB1) and anoptional first LB DNA sequence (LB1) which are positioned in the vectorto initiate (RB1) and optionally terminate (LB1) synthesis of a firstT-strand such that the first sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the vectorconfiguration further comprises a second RB DNA sequence (RB2) and anoptional second LB DNA sequence (LB2) which are positioned in the vectorto initiate (RB2) and optionally terminate (LB2) synthesis of a secondT-strand such that the first sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand. The twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other in at least a portion of the first sequenceof interest. The vector configuration further comprises a third RB DNAsequence (RB3) and an optional third LB DNA sequence (LB3) which arepositioned in the vector to initiate (RB3) and optionally terminate(LB3) synthesis of a third T-strand such that the second sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the thirdT-strand; and the vector configuration further comprises a fourth RB DNAsequence (RB4) and an optional fourth LB DNA sequence (LB4) which arepositioned in the vector to initiate (RB4) and optionally terminate(LB4) synthesis of a fourth T-strand such that the second sequence ofinterest is in an anti-sense orientation from the 5′ to 3′ end of thefourth T-strand. The two T-strands resulting from initiation at RB3 andRB4 are essentially complementary to each other in at least a portion ofthe second sequence of interest.

FIG. 3A illustrates a vector configuration where the vector comprises aRB DNA sequence and a LB DNA sequence and where the vector furthercomprises between the RB and LB DNA sequences: (i) a first sequence ofinterest in a sense orientation relative to the RB DNA sequence, (ii) aspacer, and (iii) a second sequence of interest in an anti-senseorientation relative to the RB DNA sequence. The two sequences ofinterest are essentially complementary, and after synthesis of theT-strand a hairpin structure may form. FIGS. 3B and 3C illustratepossible variations of at least two cassettes (S-o-I 1 and S-o-I 2) inrelation to positions of two homology arms within a sequence ofinterest. FIG. 3B illustrates a first cassette positioned between twohomology arms (HA) and a second cassette positioned outside of the oneof the homology arms (HA). FIG. 3C illustrates both a first cassette anda second cassette positioned between two homology arms (HA).

FIG. 4A illustrates a schematic of the control vector configuration forvector A used in Example 9.

FIG. 4B illustrates a schematic of the vector configuration for vector Cused in Example 9.

FIG. 4C illustrates a schematic of the vector configuration for vector Dused in Example 9.

FIG. 5 illustrates the targeted DNA sequence, comprising a left homologyarm (HA-L), a selectable marker gene (CP4-EPSPS), and a right homologyarm (HA-R), that is used in Example 9. FIG. 5 further illustrates theposition of PCR primers and Southern blot probes used in Example 9.

FIG. 6A illustrates a schematic of the configuration of control vector Aused in Example 12. Vector A comprises a first sequence of interestcomprising an expression cassette encoding CP4-EPSPS positioned betweena first RB DNA sequence (RB1) and a first LB DNA sequence (LB1). Thevector further comprises a second sequence of interest comprising twoexpression cassettes each encoding half of a TALEN pair positionedbetween a second RB DNA sequence (RB2) and a second LB DNA sequence(LB2).

FIG. 6B illustrates a schematic of the configuration of vector B used inExample 12. The vector comprises a first sequence of interest comprisingan expression cassette encoding CP4-EPSPS positioned between two RB DNAsequences and two LB DNA sequences. The vector further comprises asecond sequence of interest comprising two expression cassettes eachencoding half of a TALEN pair.

FIG. 7A illustrates a schematic of the configuration of vector A used inExample 13. The vector comprises a first sequence of interest comprisingan expression cassette encoding CP4-EPSPS with a TALEN target site (TS)positioned 5′ to the CP4-EPSPS cassette. Additionally, the firstsequence of interest is flanked by a first RB DNA sequence (RB1) and asecond RB DNA sequence (RB2). The vector further comprised a secondsequence of interest comprising two expression cassettes each encodinghalf of a TALEN pair positioned adjacent to a third RB DNA sequence(RB3).

FIG. 7B illustrates a schematic of the configuration of vector B used inExample 13. The vector comprises a first sequence of interest comprisingan expression cassette encoding CP4-EPSPS flanked by TALEN target sites.Additionally, the first sequence of interest is flanked by a first RBDNA sequence (RB1) and a second RB DNA sequence (RB2). The vectorfurther comprised a second sequence of interest comprising twoexpression cassettes each encoding half of a TALEN pair positionedadjacent to a third RB DNA sequence (RB3).

FIG. 7C illustrates a schematic of the configuration of vector C used inExample 13. The vector comprises a first sequence of interest comprisingan expression cassette encoding CP4-EPSPS positioned between a lefthomology arm and a right homology arm. Additionally, the first sequenceof interest is positioned between a first RB DNA sequence (RB1) and asecond RB DNA sequence (RB2). The vector further comprises a secondsequence of interest comprising two expression cassettes each encodinghalf of a TALEN pair positioned adjacent to a third RB DNA sequence(RB3).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used havethe same meaning as commonly understood by one of ordinary skill in theart to which this disclosure belongs. Where a term is provided in thesingular, the inventors also contemplate aspects of the disclosuredescribed by the plural of that term. Where there are discrepancies interms and definitions used in references that are incorporated byreference, the terms used in this application shall have the definitionsgiven herein. Other technical terms used have their ordinary meaning inthe art in which they are used, as exemplified by various art-specificdictionaries, for example, “The American Heritage® Science Dictionary”(Editors of the American Heritage Dictionaries, 2011, Houghton MifflinHarcourt, Boston and New York), the “McGraw-Hill Dictionary ofScientific and Technical Terms” (6th edition, 2002, McGraw-Hill, NewYork), or the “Oxford Dictionary of Biology” (6th edition, 2008, OxfordUniversity Press, Oxford and New York). The inventors do not intend tobe limited to a mechanism or mode of action. Reference thereto isprovided for illustrative purposes only.

The practice of this disclosure includes, unless otherwise indicated,conventional techniques of biochemistry, chemistry, molecular biology,microbiology, cell biology, genomics and biotechnology, which are withinthe skill of the art. See Green and Sambrook, Molecular Cloning: ALaboratory Manual, 4th edition (2012); Current Protocols In MolecularBiology (F. M. Ausubel, et al. eds., (1987)); the series Methods InEnzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J.MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Harlow and Lane,eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (R. I.Freshney, ed. (1987)); Recombinant Protein Purification: Principles AndMethods, 18-1142-75, GE Healthcare Life Sciences; C. N. Stewart, A.Touraev, V. Citovsky, T. Tzfira eds. (2011) Plant TransformationTechnologies (Wiley-Blackwell); and R. H. Smith (2013) Plant TissueCulture: Techniques and Experiments (Academic Press, Inc.).

All references cited herein are incorporated by reference in theirentireties.

As used herein, terms in the singular and the singular forms “a,” “an,”and “the,” for example, include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “plant,”“the plant,” or “a plant” also includes a plurality of plants; also,depending on the context, use of the term “plant” can also includegenetically similar or identical progeny of that plant; use of the term“a nucleic acid” optionally includes, as a practical matter, many copiesof that nucleic acid molecule; similarly, the term “probe” optionally(and typically) encompasses many similar or identical probe molecules.

As used herein, “plant” refers to a whole plant. A cell or tissueculture derived from a plant can comprise any plant components or plantorgans (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plantcells, and/or progeny of the same. A progeny plant can be from anyfilial generation, e.g., F₁, F₂, F₃, F₄, F₅, F₆, F₇, etc. A plant cellis a biological cell of a plant, taken from a plant or derived throughculture from a cell taken from a plant.

As used herein, “transgenic” means a plant cell, a plant, a plant part,or a seed whose genome has been altered by the stable integration ofexogenous DNA. A transgenic line includes a plant regenerated from anoriginally-transformed plant cell and progeny transgenic plants fromlater generations or crosses of a transformed plant.

As used herein, “stably transformed” is defined as a transfer of DNAinto genomic DNA of a targeted cell that allows the targeted cell topass the transferred DNA to the next generation. In some embodiments thetransferred DNA is integrated into the genomic DNA of a reproductivecell. In some embodiments the transferred DNA is integrated into thegenomic DNA of a somatic cell. As used herein, “transiently transformed”is defined as a transfer of DNA into a cell that is not integrated intothe transformed cell's genomic DNA.

As used herein, “plant genome” refers to a nuclear genome, amitochondrial genome, or a plastid (e.g., chloroplast) genome of a plantcell.

In one aspect, the instant disclosure provides a Rhizobiales cellcomprising at least one vector that is capable of forming at least twoT-strands that are essentially complementary in at least a portion of asequence of interest. In another aspect, the instant disclosure providesa Rhizobiales cell comprising at least one vector that is capable offorming at least two T-strands that are essentially complementary for atleast a portion of the vector backbone. In another aspect, the instantdisclosure provides a Rhizobiales cell comprising at least one vectorthat is capable of forming two T-strands that are essentiallycomplementary for at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300,2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300,3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300,4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300,5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300,6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000 7,100, 7,200, 7,300,7,400, 7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300,8,400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300,9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000, or more base pairs. Insome aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more vectors providedherein are located within a single Rhizobiales cell.

In another aspect, the instant disclosure provides a Rhizobiales cellcomprising at least a first vector and a second vector, where the firstvector is configured to produce a first T-strand comprising a sequenceof interest and the second vector is configured to produce a secondT-strand comprising a sequence of interest oriented such that the firstand second T-strands are essentially complementary in at least a portionof a sequence of interest. In another aspect, the instant disclosureprovides a Rhizobiales cell comprising at least a first vector and asecond vector that are capable of producing a first T-strand and asecond T-strand that are essentially complementary for at least aportion of the vector backbone. In another aspect, the instantdisclosure provides a Rhizobiales cell comprising at least a firstvector and a second vector that are capable of producing a firstT-strand and a second T-strand that are essentially complementary for atleast 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600,2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600,3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600,4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600,5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600,6,700, 6,800, 6,900, 7,000 7,100, 7,200, 7,300, 7,400, 7,500, 7,600,7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600,8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600,9,700, 9,800, 9,900, 10,000, or more base pairs. In some aspects, 3, 4,5, 6, 7, 8, 9 or 10 or more vectors provided herein are located within asingle Rhizobiales cell.

As used herein, the term “Rhizobiales” refers to members of thebacterial Order Rhizobiales that are capable of transforming a plantcell. In some aspects, a Rhizobiales provided herein can refer to anAgrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., anOchrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and aSinorhizobium spp. In other aspects, a Rhizobiales provided herein canrefer to Agrobacterium tumefaciens or Agrobacterium rhizogenes. In someaspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more vectors provided hereinare located within a single Rhizobiales cell. In other aspects, 2, 3, 4,5, 6, 7, 8, 9 or 10 or more different vectors provided herein arelocated within 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more differentRhizobiales cells.

As used herein, the term “recombination” refers to the exchange ofnucleotides between two nucleic acid molecules. The term “homologousrecombination” (HR) refers to the exchange of nucleotides at a conservedregion shared by two nucleic acid molecules. HR includes symmetrichomologous recombination and asymmetric homologous recombination.Asymmetric homologous recombination can also mean unequal recombination.As used herein, “non-homologous end joining” (NHEJ) refers to theligation of two ends of double-stranded DNA without the need of ahomologous sequence to direct the ligation. As used herein,“microhomology” refers to the presence of the same short sequence (1 to10 bp) of bases in different nucleic acid molecules. In someembodiments, at least one of the nucleic acid molecules is genomic DNAand at least one of the nucleic acid molecules comprises two T-strandsthat are essentially complementary in at least a portion of a sequenceof interest.

Methods for detecting HR and NHEJ include, but are not limited to, 1)phenotypic screening, 2) molecular marker technologies such as singlenucleotide polymorphism (SNP) analysis by TaqMan® or Illumina/Infiniumtechnology, 3) Southern blot, and 4) sequencing (e.g., Sanger,Illumina®, 454, Pac-Bio, Ion Torrent™). One example of a method foridentifying recombination between two parental chromosomes is inversePCR (iPCR). In this method, restriction nuclease sites flanking atargeted gene are identified on each of the two parental chromosomes.These restriction nuclease sites can be the same or different. A PCRprimer specific for the first parental chromosome and another PCR primerspecific for the second parental chromosome are designed. An induceddouble-strand break promotes recombination between the two parentalchromosomes brings both restriction endonuclease sites and primerbinding sites onto the same recombinant chromosome. A PCR product isobserved only in instances where recombination occurs. In one aspect,the occurrence of homologous recombination can be detected by PCR, withprimers specifically designed for the T-strand insert paired withprimers specifically designed for flanking sequences of the targetsequence (outside of the homologous regions). For example, when anupstream flanking primer is paired with a downstream insert-specificprimer, a PCR product is observed only when recombination occurs.

As used herein, the term “vector” or “plasmid” is used interchangeablyand refer to a circular, double-stranded DNA molecule that is physicallyseparate from chromosomal DNA. In one aspect, a plasmid or vector usedherein is capable of replication in vivo. In several embodiments, thevector is capable of transforming a plant cell. In an aspect, a plasmidprovided herein is a bacterial plasmid. In another aspect, a plasmidprovided herein is an Agrobacterium Ti plasmid or derived from anAgrobacterium Ti plasmid.

In one aspect, a plasmid or vector provided herein is a recombinantvector. As used herein, the term “recombinant vector” refers to a vectorformed by laboratory methods of genetic recombination, such as molecularcloning. In another aspect, a vector or plasmid provided herein is asynthetic plasmid. As used herein, a “synthetic plasmid” is anartificially created plasmid that is capable of the same functions(e.g., replication) as a natural plasmid (e.g., Ti plasmid). Withoutbeing limited, one skilled in the art can create a synthetic plasmid denovo via synthesizing a plasmid by individual nucleotides, or bysplicing together nucleic acid molecules from different pre-existingplasmids.

As used herein, the terms “homology” and “identity” when used inrelation to nucleic acids, describe the degree of similarity between twoor more nucleotide sequences. The percentage of “sequence identity”between two sequences is determined by comparing two optimally alignedsequences over a comparison window, such that the portion of thesequence in the comparison window may comprise additions or deletions(gaps) as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity. A sequence that is identical at every position incomparison to a reference sequence is said to be identical to thereference sequence and vice-versa. An alignment of two or more sequencesmay be performed using any suitable computer program. For example, awidely used and accepted computer program for performing sequencealignments is CLUSTALW v1.6 (Thompson, et al. (1994) Nucl. Acids Res.,22: 4673-4680).

As used herein, the term “complementary” in reference to a nucleic acidmolecule refers to pairing of nucleotide bases such that A iscomplementary to T (or U), and G is complementary to C. Twocomplementary nucleic acid molecules are capable of hybridizing witheach other. As an example, the two strands of double stranded DNA arecomplementary to each other.

As used herein, the term “essentially homologous” or “essentiallyidentical” means that two nucleotide sequences have at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity with each other.

As used herein, the term “essentially complementary” means that twonucleotide sequences have at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence complementarity with eachother.

As used herein, a “portion” of a nucleic acid molecule refers to atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or 100% of its total length, or at least 5, 10, 15, 20, 25,30, 25, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, or 1000 or more contiguous nucleotides.

As used herein, the term “polynucleotide” refers to a nucleic acidmolecule containing multiple nucleotides and generally comprises atleast 2, at least 5, at least 10, at least 20, at least 30, at least 40,at least 50, at least 100, at least 250, at least 500, at least 1000, atleast 1500, at least 2000, at least 2500, at least 3000, at least 5000,at least 10,000 or more nucleotide bases. As an example, apolynucleotide provided herein can be a plasmid. A specificpolynucleotide of 18-25 nucleotides in length may be referred to as an“oligonucleotide”. Nucleic acid molecules provided herein includedeoxyribonucleic acids (DNA) and ribonucleic acids (RNA) and functionalanalogues thereof, such as complementary DNA (cDNA). Nucleic acidmolecules provided herein can be single stranded or double stranded.Nucleic acid molecules comprise the nucleotide bases adenine (A),guanine (G), thymine (T), cytosine (C). Uracil (U) replaces thymine inRNA molecules. The symbol “N” can be used to represent any nucleotidebase (e.g., A, G, C, T, or U). The symbol “K” can be used to represent aG or a T/U nucleotide base.

As used herein, “physically linked” means that the physically linkednucleic acid sequences are located on the same nucleic acid molecule. Aphysical linkage can be adjacent or proximal. In an aspect, a nucleicacid sequence provided herein is adjacent to another nucleic acidsequence. In another aspect, a first nucleic acid molecule providedherein is physically linked to a second nucleic acid molecule providedherein. As used herein, “flanked” refers to a nucleic acid sequence thatis linked on one or both sides to another nucleic acid sequence,including linked to a sequence of interest, or to a LB DNA sequence, ora RB DNA sequence. In one aspect, a flanking sequence precedes asequence of interest. In another aspect, a flanking sequence follows asequence of interest. In one aspect, a sequence of interest is flankedby another sequence of interest. In yet another aspect, a flankingsequence is on the 5′ or upstream end of a sequence of interest. Inanother aspect, a flanking sequence is on the 3′ or downstream end of asequence of interest. In some embodiments, the flanking sequence iscontiguous with the sequence of interest. In some embodiments, there areone or more nucleotides between the flanking sequence and the sequenceof interest.

As used herein, “operably linked” means that the operably linked nucleicacid sequences exhibit their desired function. For example, in an aspectof this disclosure, a provided DNA promoter sequence can initiatetranscription of an operably linked DNA sequence into RNA. A nucleicacid sequence provided herein can be upstream or downstream of aphysically or operably linked nucleic acid sequence. In an aspect, afirst nucleic acid molecule provided herein is both physically linkedand operably linked to a second nucleic acid molecule provided herein.In another aspect, a first nucleic acid molecule provided herein isneither physically linked nor operably linked to a second nucleic acidmolecule provided herein. As used herein, “upstream” means the nucleicacid sequence is positioned before the 5′ end of a linked nucleic acidsequence. As used herein, “downstream” means the nucleic acid sequenceis positioned after the 3′ end of a linked nucleic acid sequence.

As used herein, a “spacer” or a “linker” refers to any polynucleotidesequence capable of forming a loop structure that is at least 4nucleotides in length. Without being limiting, a spacer provided hereincan comprise a non-coding sequence or a coding sequence.

Right and Left Borders

As used herein, the term “T-strand” refers to a single-strand copy ofDNA made when transcription is initiated from a RB DNA sequence of avector. In a naturally occurring Ti plasmid, synthesis of the transferDNA (T-DNA) is initiated at a 25-base-pair consensus DNA sequencereferred to as the “right border” (RB), and T-strand synthesistermination occurs at a 25 bp consensus DNA sequence referred to as the“left border” (LB). The 25 bp RB consensus DNA sequence (SEQ ID NO: 21)and the 25 bp LB consensus DNA sequence (SEQ ID NO: 23) are from thenopaline Agrobacterium tumefaciens strain C58. The 25 bp RB consensusDNA sequence (SEQ ID NO: 22) and the 25 bp LB consensus DNA sequence(SEQ ID NO: 24) are from the octopine Agrobacterium tumefaciens strainA6. In some embodiments, a RB consensus DNA sequence is selected fromSEQ ID NOs: 21 and 23. In some embodiments a LB consensus DNA sequenceis selected from SEQ ID NOs: 22 and 24.

In one aspect, a RB DNA sequence or LB DNA sequence provided hereincomprises at least a 25 bp Ti plasmid RB or LB (respectively) consensusDNA sequence and may additionally comprise a nucleic acid sequencecomprising at least 25 nucleotides, at least 50 nucleotides, at least100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, atleast 250 nucleotides, at least 300 nucleotides, at least 350nucleotides, at least 400 nucleotides, at least 450 nucleotides, atleast 500 nucleotides, or at least 600 nucleotides. In an aspect, a RBDNA sequence or a LB DNA sequence provided herein can be of any lengthsuch that the DNA segment is capable of transformation of plant tissueperformed by Agrobacterium or other Rhizobiales-mediated methods (SeeU.S. Pat. Nos. 5,731,179 and 6,265,638; and U.S. Patent ApplicationPublications US2003/110532; US2005/0183170; and US2007/0271627).

As used in this application, RB DNA sequences are presented as SEQ IDNOs: 1-13 and are variants of A. tumefaciens Ti plasmid sequencesranging from 162 nt to 505 nt in length and comprising either thenopaline RB consensus DNA sequence (SEQ ID NO: 21) or the octopine RBconsensus DNA sequence (SEQ ID NO: 22). Similarly, as used in thisapplication, LB DNA sequences are presented as SEQ ID NOs: 14-20 and arevariants of A. tumefaciens Ti plasmid sequences ranging from 220 nt to443 nt in length and comprising either the nopaline LB consensus DNAsequence (SEQ ID NO: 23) or the octopine LB consensus DNA sequence (SEQID NO: 24). Table 1 contains the SEQ ID NOs for each of the RB and LBDNA sequences presented herein with the position indicated within eachSEQ ID NO of the nopaline or octopine 25 bp consensus sequences. OtherRB and LB sequences are contemplated.

TABLE 1 SEQ ID NOs of disclosed right border (RB) and left border (LB)DNA sequences RB or LB Nopaline Position SEQ ID Length DNA or of 25 bpNO (# of nt) Sequence Octopine consensus 1 355 RB Nopaline 291-315 2 356RB Nopaline 292-316 3 355 RB Nopaline 291-315 4 505 RB Octopine 194-2185 470 RB Nopaline 293-317 6 334 RB Octopine 194-218 7 162 RB Nopaline 98-122 8 329 RB Nopaline 291-315 9 331 RB Nopaline 293-317 10 331 RBNopaline 293-317 11 285 RB Octopine 194-218 12 357 RB Nopaline 293-31713 330 RB Nopaline 292-316 14 220 LB Nopaline 46-70 15 401 LB Octopine222-246 16 443 LB Octopine 263-287 17 319 LB Nopaline 58-82 18 427 LBNopaline 166-190 19 442 LB Octopine 263-287 20 411 LB Octopine 232-256

In one aspect, a vector provided herein can comprise at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10 RB DNA sequences. In one aspect ofthe vector, the RB DNA sequence comprises an octopine Agrobacterium Tiplasmid 25 bp RB DNA consensus sequence. In another aspect of thevector, the RB DNA sequence comprises a nopaline Agrobacterium Tiplasmid 25 bp RB consensus DNA sequence. In another aspect, in vectorconfigurations with two or more RB DNA sequences, the RB DNA sequencesmay be essentially homologous or the RB DNA sequences may not beessentially homologous. In another aspect, the one or more RB DNAsequences comprise a sequence at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% identical, or 100% identical to a sequenceselected from SEQ ID NOs: 1-13. In another embodiment, the first RB(RB1) DNA sequence or the second RB (RB2) DNA sequence comprises asequence selected from the group comprising SEQ ID NOs: 1-13. In someembodiments, the RB1 DNA sequence comprises a sequence at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identical, or 100%identical to a sequence selected from SEQ ID NO: 4 or SEQ ID NO: 12; andthe RB2 DNA sequence comprises a sequence at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identical, or 100% identical to asequence selected from SEQ ID NO: 4 or SEQ ID NO: 12.

In one aspect, the at least one vector disclosed herein does notcomprise a LB DNA sequence. In another aspect, the at least one vectordisclosed herein comprises at least one, at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 LB DNA sequences. In another aspect, in vectorconfigurations with two or more LB DNA sequences, the LB DNA sequencesmay be essentially homologous or the LB DNA sequences may not beessentially homologous. In a further aspect, the LB DNA sequencecomprises an octopine Agrobacterium Ti plasmid 25 bp LB consensus DNAsequence. In another aspect, the LB DNA sequence comprises a nopalineAgrobacterium Ti plasmid 25 bp LB consensus DNA sequence. In one aspect,the one or more of the LB DNA sequences are selected from the groupcomprising SEQ ID NOs: 14-20. In another aspect, the two or more LB DNAsequences comprise a sequence at least 80%, at least 81%, at least 81%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to a sequence selected fromSEQ ID NOs: 14-20. In one aspect, the LB DNA sequence comprises asequence at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identical, or 100% identical to SEQ ID NO: 19.

Sequence of Interest

As used herein, the term “sequence of interest” refers to apolynucleotide sequence in a vector that forms part of the T-strand. Inone aspect, a vector provided herein comprises 0, at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7, at least8, at least 9, or at least 10, or more sequences of interest. As usedherein, a sequence of interest in a vector disclosed herein does notinclude RB or a LB DNA sequences, and does not include vector backbonesequence. In one aspect, in vector configurations with two sequences ofinterest positioned between two RB DNA sequences such that the sequenceof interest in the two T-strands synthesized from the two RB DNAsequences are essentially complementary. In some embodiments, thenucleotide sequence of the two sequences of interest may be identical,or the nucleotide sequence may be at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical.

In some aspects, a sequence of interest provided herein comprises 0, atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10 expression cassettes. Insome aspects, the sequence of interest provided herein comprises one ormore expression cassettes physically and/or operably linked in acassette stack. In some aspects a sequence of interest comprises anexpression cassette adjacent to a left homology arm DNA sequence, aright homology arm DNA sequence, or a left homology arm DNA sequence anda right homology arm DNA sequence. In some aspects, a sequence ofinterest comprises an expression cassette flanked by homology arm DNAsequences. In some aspects, a sequence of interest comprises one oremore expression cassettes that is not flanked by homology arms. In someaspects, a sequence of interest comprises one or more expressioncassettes flanked by one ore more site-specific enzyme target sites. Insome aspects, a sequence of interest comprises one or more expressioncassettes flanked by one ore more recombinase recognition sites. Inanother aspect, a sequence of interest provided herein comprises anendogenous polynucleotide sequence. In some embodiments, the endogenouspolynucleotide sequence comprises an intergenic sequence, a native gene,or a mutated gene. In another aspect, a sequence of interest providedherein comprises an exogenous polynucleotide sequence.

In one aspect, at least part of the sequence of interest in the vectordisclosed herein is integrated into a plant genome via HR. In anotheraspect, at least part of the sequence of interest in the vectordisclosed herein is integrated into a plant genome via NHEJ.

In one aspect, a sequence of interest is flanked by at least twohomology arm DNA sequences and comprises a sequence that is at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a native gene of a plant cell. In some embodiments, the atleast two homology are sequences are flanked by one or moresite-specific enzyme target sites. In some embodiments, the at least twohomology are sequences are flanked by one or more recombinaserecognition sites.

Homology Arms

In an aspect, a sequence of interest provided herein comprises 0, atleast 1, or at least 2 homology arm DNA sequences. When a sequence ofinterest provided herein comprises at least two homology arm DNAsequences the at least two homology arm DNA sequences can bedistinguished by referring to them as a “left homology arm DNA sequence”and a “right homology arm DNA sequence.” In an aspect, a sequence ofinterest provided herein comprises both a left homology arm DNA sequenceand a right homology arm DNA sequence. In an aspect, a right homologyarm DNA sequence and a left homology arm DNA sequence provided hereinare homologous to a targeted genomic DNA sequence in the plant or plantcell. In an aspect, a right homology arm DNA sequence and a lefthomology arm DNA sequence are not essentially homologous to each other.In another aspect, a right homology arm DNA sequence and a left homologyarm DNA sequence are essentially homologous to each other. In an aspect,a sequence of interest comprises one or more expression cassettespositioned between a right homology arm DNA sequence and a left homologyarm DNA sequence. In an aspect, a sequence of interest comprises asequence for templated genome editing positioned between a righthomology arm DNA sequence and a left homology arm DNA sequence. In yetanother aspect, at least part of a sequence of interest provided hereinis outside of the region comprising a left homology arm DNA sequence, aright homology arm DNA sequence, and one or more cassettes. In anotheraspect, at least part of a sequence of interest provided herein iswithin the region comprising a left homology arm DNA sequence, a righthomology arm DNA sequence, and a sequence for templated genome editing.

In one aspect, a sequence of interest provided herein comprises a firstsequence positioned between a left homology arm DNA sequence and a righthomology arm DNA sequence, and a second sequences that is not positionedbetween a left homology arm DNA sequence and a right homology arm DNAsequence. In another aspect, a vector provided herein comprises a firstsequence of interest further comprising a first left homology arm DNAsequence and a first right homology arm DNA sequence and a secondsequence of interest further comprising a second left homology arm DNAsequence and a second right homology arm DNA sequence.

In an aspect, a sequence of interest provided herein is integrated intoa plant genome in its entirety. In an aspect, at least a part of asequence of interest provided herein is integrated into a plant genome.In another aspect, only the sequence of interest between a righthomology arm DNA sequence and a left homology arm DNA sequence isintegrated into a plant genome. In another aspect, a sequence ofinterest provided herein is integrated into a plant genome via HR. In anaspect, HR can occur between one or two homology arm DNA sequencesprovided herein and a plant genome. In another aspect, a sequence ofinterest provided herein is integrated into a plant genome via NHEJ. Inanother aspect, a sequence of interest provided herein is integratedinto a plant genome via microhomology-mediated end joining. In yetanother aspect, a sequence of interest provided herein is randomlyintegrated into a plant genome via Agrobacterium-mediated T-strandintegration.

In an aspect, integration of at least part of a sequence of interestprovided herein results in one or more point mutations, one or moreinsertions, one or more deletions, an inversion, increased transcriptionof an endogenous locus, decreased transcription of an endogenous locusor any combination thereof. In an aspect, integration of at least partof a sequence of interest provided herein results in altered proteinactivity, altered production of RNAi polynucleotides, altered sequenceof RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, silencing of the integratedsequence of interest, or any combination thereof. Silencing technologiesinclude, without limitations, antisense-, co-suppression-mediatedmechanisms, and RNAi technologies, such as miRNA (e.g., U.S. PatentApplication Publication 2006/0200878). In another aspect, integration ofat least part of a sequence of interest causes targeted transcription,decreased transcription, enhanced transcription, or templated editing ofa transgenic nucleic acid sequence present in the genome.

As used herein, the term “homology arm” or “homology arm DNA sequence”refers to a polynucleotide sequence that has at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to a target sequence in a plant or plant cell. Ahomology arm can comprise at least 5, at least 10, at least 15, at least20, at least 25, at least 30, at least 40, at least 50, at least 100, atleast 150, at least 200, at least 250, at least 300, at least 350, atleast 400, at least 450, at least 500, at least 500, at least 550, atleast 600, at least 650, at least 700, at least, 750, at least 800, atleast 850, at least 900, at least 950, at least 1000, or at least 2500nucleotides. In one aspect, the target sequence comprises aprotein-coding sequence. In one aspect, the target sequence is a genicsequence. As used herein, a “genic” sequence is a nucleic acid sequencethat encodes a protein or a non-protein-coding RNA. A genic sequence caninclude one or more introns. In another aspect, the target sequence is anon-genic sequence. As used herein, a “non-genic” sequence is a nucleicacid sequence that is not a genic sequence. In another aspect, thetarget sequence comprises a non-coding sequence. In yet another aspect,the target sequence comprises both a protein-coding sequence and anon-coding sequence. In another aspect, the target sequence does notcomprise a gene or a portion of a gene. In some embodiments, the targetsequence is linked to a gene of interest. In some embodiments, thetarget sequence is linked to a transgene integrated in the genome of aplant or plant cell.

As used herein, the term “target sequence” refers to a genomic locusselected for targeted integration of a sequence of interest. In someembodiments, the sequence of interest is integrated into the targetsequence by HR or NHEJ. Depending upon the circumstances, the termtarget sequence can refer to the full-length nucleotide sequence of thegenomic locus targeted for cleavage and recombination, or the nucleotidesequence of a portion of the genomic locus targeted for cleavage andrecombination. The target sequence can be an endogenous genomic locus ora transgene. In one aspect, a target sequence is a genic sequence. Inanother aspect, a target sequence is a non-genic sequence.

As used herein, a “site-specific recombination site” or “site-specifictarget site” are used interchangeably to refer to a nucleic acidsequence where exogenous DNA is inserted by HR or by non-homologousrecombination.

As used herein “site-specific enzyme target site” refers to the sitethat is cleaved by a nuclease introducing a double stranded break intothe nucleic acid backbone. The site of the double-strand break may be atarget site for introduction of a sequence of interest.

As used herein, “endogenous” refers to a nucleic acid sequence thatexists naturally in the genome of a cell. In an aspect, a methodprovided herein is used to modify an endogenous locus so that themodified locus shares at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or at least 99.9% sequence identityas compared to an unmodified endogenous locus. In one aspect, anendogenous nucleic acid sequence undergoes “template editing.” Templateediting can occur via HR between a target sequence and a donor templateafter a double-stranded break occurs in or near the target sequence. Asused herein, a “donor template” is a nucleic acid sequence that sharesat least 60% at least 60%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or at least 99.9% sequenceidentity to at least a portion of a target sequence. In one aspect, adonor template provided herein comprises a sequence of interest.Template editing can introduce one or more point mutations, deletions,or insertions into a target sequence. In one aspect, the entire donortemplate, a portion of the donor template, a copy of the donor template,or a portion of a copy of the donor template integrates into the targetsequence. One of ordinary skill in the art would recognize that suchtemplate editing would be analogous to an orthologous nucleic acidsequence, a paralogous nucleic acid sequence, an isogenic nucleic acidsequence, or a cisgenic nucleic acid sequence of the endogenous genome.

As used herein “exogenous” refers to a nucleic acid sequence that is notnormally present in a cell, but can be introduced into a cell by one ormore genetic, biochemical or other methods. In some embodiments, anexogenous nucleic acid sequence can be homologous to an endogenousmolecule.

Cassettes

As used herein, the terms “cassette” or “expression cassette” refer to anucleic acid sequence which may or may not be operably linked to one ormore expression elements such as an enhancer, a promoter, a leader, anintron, a 5′ untranslated region (UTR), a 3′ UTR, or a transcriptiontermination sequence. In one aspect, a cassette comprises a nucleic acidsequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to an endogenous nucleic acid sequence. Inanother aspect, the cassette comprises an exogenous nucleic acidsequence.

In one aspect, a cassette in a vector disclosed herein comprises atleast one sequence selected from: a gene, a portion of a gene, anintergenic sequence, an enhancer, a promoter, an intron, an exon, atranscription termination sequence, a sequence encoding a chloroplasttargeting peptide, a sequence encoding a mitochondrial targetingpeptide, an insulator sequence, a sequence encoding an anti-sense RNA, asequence encoding non-protein-coding RNA (npcRNA), a sequence encoding arecombinase, a sequence encoding a recombinase recognition site (forexample, a lox site or a flp site), a sequence encoding an Argonaute(non-limiting examples of Argonaute proteins include Thermusthermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo),Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, ormodified versions thereof), a sequence encoding DNA guide, a sequenceencoding an RNA-guided endonuclease (non-limiting examples of RNA-guidednucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8,Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1,Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3,Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX,Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, homologsthereof, or modified versions thereof), a sequence encoding a CRISPRRNA, a sequence encoding a tracrRNA, a sequence encoding a fused guideRNA, a sequence encoding a zinc finger nuclease, a sequence encoding aTALEN, a landing pad, an editing template, a transgene, a sequenceencoding a site-specific enzyme, a sequence encoding a site-specificenzyme target site, a sequence encoding a selection marker, a sequenceencoding a cell factor that functions to increase DNA repair, a linker,a spacer, a restriction enzyme site, a sequence for templated genomeediting, and any combination thereof. In some embodiments, the cassetteis positioned between a first right border (RB1) and a second rightborder (RB2) such that the two T-strands resulting from initiation atRB1 and RB2 are essentially complementary to each other in at least aportion of the cassette. In some embodiments, the cassette is positionedadjacent to at least one LB DNA sequence. In some embodiments, thecassette is not positioned between RB DNA sequences. In someembodiments, the cassette is not positioned between a RB DNA sequenceand a LB DNA sequence. As used herein, the term “landing pad” refers toa nucleic acid locus that is designed to be the locus for site-specificrecombination. In some embodiments, landing pads may comprise one ormore nucleic acid sequences that are not homologous to native sequencesof the host organism. In some embodiments, landing pads may comprise oneor more recognition sites for any of an endonuclease, a recombinase or atransposase. In some embodiments a landing pad may comprise one or morerecognition sites for any of an endonuclease, a recombinase or atransposase flanking one or more nucleotide sequences substantiallylacking homology with the genome of the host organism. In someembodiments, the one or more recognition sites are binding sites forDNA-binding domains (e.g., zinc finger proteins (ZFPs), meganucleases,or leucine zippers). In some examples, landing pads may comprisenucleotide sequences that have substantially no homology to regions ofany sequenced target plant genome. In some embodiments, the landing padmay comprises any combination of one or more Zinc Finger Nucleaserecognition sites, one or more meganuclease recognition sites, one ormore targeting endonuclease recognition sites, one or more TALENrecognition sites, one or more recombinase recognition sites, or one ormore transposase recognition sites. As used herein, the term “editingtemplate” refers to a nucleic acid sequence that can be used forrecombination with a target sequence. In one aspect, an editing templatecomprises a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least 99% identical to at least a portionof a target sequence. In some embodiments, the editing templatecomprises one or more nucleic acid changes compared to the targetsequence.

As used herein, the term “cassette stack” refers to two or moreexpression cassettes which are physically linked in a vector. In someembodiments, two or more cassettes in a cassette stack are operablylinked. In some embodiments, two or more cassettes in a cassette stackare not operably linked. In some embodiments, two or more cassettes in acassette stack are flanked by one or more recombinase recognition sites.In some embodiments, two or more cassettes in a cassette stack areflanked by one or more a site-specific enzyme target sites. In someembodiments, two or more cassettes in a cassette stack may be separatedby spacer sequences, insulator sequences, multiple cloning sitesequences, one or more recombinase recognition sites, a sequenceencoding a site-specific enzyme target site, a landing pad, homologyarms, a RB DNA sequence or a LB DNA sequence. In some embodiments, thecassette stack is positioned between a first right border (RB1) and asecond right border (RB2) such that the two T-strands resulting frominitiation at RB1 and RB2 are essentially complementary to each other inat least a portion of the cassette stack. In some embodiments, thecassette stack is positioned adjacent to at least one LB DNA sequence.In some embodiments, the cassette stack is not positioned between RB DNAsequences. In some embodiments, the cassette stack is not positionedbetween a RB DNA sequence and a LB DNA sequence.

As used herein, the term “genomic locus” means a locatable region ofgenomic sequence, corresponding to a unit of inheritance. A genomiclocus may comprise one or more regulatory regions, such as promoters,enhancers, 5′ UTRs, intron regions, 3′UTRs, transcribed regions, andother functional sequence regions that may exist as native genes ortransgenes in a plant or a mammalian genome.

As used herein, “gene” refers to a sequence that encodes a protein, or asequence encoding a non-protein-coding RNA. As used herein,“protein-coding” refers to a polynucleotide encoding for the amino acidsof a polypeptide. As used herein, “encoding” refers to a polynucleotidethat can produce a functional unit (without being limiting, for example,a protein, a microRNA, a transfer RNA, a ribosomal RNA, a smallinterfering RNA, a guide RNA, a tracer RNA, a single-guide RNA) viatranscription and/or translation.

A series of three nucleotide bases encodes one amino acid. As usedherein, “expressed,” “expression,” or “expressing” refers totranscription of RNA from a DNA molecule. In one aspect, a sequence ofinterest provided herein comprises a protein-coding sequence.

As used herein, the term “npcRNA” refers to non-protein-coding RNA.Non-limiting examples of non-protein-coding RNA include a microRNA(miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA(22-26 nt in length) and precursor encoding same, a heterochromaticsiRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin doublestrand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturallyoccurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA(gRNA), and a single-guide RNA (sgRNA). In one aspect, a sequence ofinterest provided herein comprises a non-protein-coding RNA.

In one aspect, an expression cassette comprises at least one geneselected from a gene of agronomic interest, a DNA binding gene, aselectable marker gene, an RNAi construct, a site specific nucleasegene, a recombinant guide RNA of an RNA-guided nuclease (non-limitingexamples of RNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4,Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10,Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4,Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,Cpf1, CasX, CasY, homologs thereof, or modified versions thereof), anArgonaute (non-limiting examples of Argonaute proteins include Thermusthermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo),Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, modifiedversions thereof), a DNA guide for an Argonaute protein, and anycombination thereof. In an aspect, a gene provided herein comprises apromoter. In another aspect, a gene provided herein does not comprise apromoter.

Examples of suitable genes of agronomic interest envisioned by thisdisclosure would include but are not limited to genes for disease,insect, or pest tolerance (for example, virus tolerance, bacteriatolerance, fungus tolerance, nematode tolerance, arthropod tolerance,gastropod tolerance), herbicide tolerance, genes for qualityimprovements such as yield, nutritional enhancements, environmental orstress tolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s) including starch production(U.S. Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295),modified oils production (U.S. Pat. Nos. 6,444,876; 6,426,447;6,380,462), high oil production (U.S. Pat. Nos. 6,495,739; 5,608,149;6,483,008; 6,476,295), modified fatty acid content (U.S. Pat. Nos.6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538;6,589,767; 6,537,750; 6,489,461; 6,459,018), high protein production(U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466),enhanced animal and human nutrition (U.S. Pat. Nos. 6,723,837;6,653,530; 6,541,259; 5,985,605; 6,171,640), biopolymers (U.S. Pat. Nos.RE37,543; 6,228,623; 5,958,745 and U.S. Patent Publication No.US20030028917). Also environmental stress resistance (U.S. Pat. No.6,072,103), pharmaceutical peptides and secretable peptides (U.S. Pat.Nos. 6,812,379; 6,774,283; 6,140,075; 6,080,560), improved processingtraits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzymeproduction (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seedproduction (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos.6,576,818; 6,271,443; 5,981,834; 5,869,720) and biofuel production (U.S.Pat. No. 5,998,700). Any of these or other genetic elements, methods,and transgenes can be used with the disclosure as will be appreciated bythose of skill in the art in view of this disclosure.

In one aspect, an expression cassette provided herein comprises anucleic acid sequence selected from an insecticidal resistance gene, anherbicide tolerance gene, a nitrogen use efficiency gene, a water useefficiency gene, a nutritional quality gene, a DNA binding gene, aselectable marker gene, an RNAi construct, a site specific nucleasegene, a guide RNA, and any combination thereof.

A gene can also include polynucleotide sequences that encode for otherpolynucleotide sequences such as a messenger RNA (mRNA). An mRNAproduced from a nucleic acid molecule of this disclosure can contain a5′-UTR leader sequence. This sequence can be derived from the promoterselected to express the gene and can be specifically modified so as toincrease or decrease translation of the mRNA. A 5′-UTR can also beobtained from viral RNAs, from suitable eukaryotic genes, or from asynthetic gene sequence. Such “enhancer” sequences can be desirable toincrease or alter the translational efficiency of the resultant mRNA.This disclosure is not limited to constructs where the non-translatedregion is derived from the 5′-UTR that accompanies the promotersequence. Rather, the 5′-UTR sequence can be derived from unrelatedpromoters or genes (see, for example U.S. Pat. No. 5,362,865). Examplesof non-translation leader sequences include maize and petunia heat shockprotein leaders (U.S. Pat. No. 5,362,865), plant virus coat proteinleaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK(U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and Freed, Journal ofVirology, (1990) 64: 1590-1597), and AGRtu.nos (GenBank AccessionV00087; Bevan et al., Nucleic Acids Research (1983) 11:369-385). Othergenetic components that serve to enhance expression or affecttranscription or translational of a gene are also envisioned as geneticcomponents.

A gene can further comprise a 3′-UTR. The provided 3′-UTRs can contain atranscriptional terminator, or an element having equivalent function,and a polyadenylation signal that functions in plants to cause theaddition of polyadenylated nucleotides to the 3′ end of an RNA molecule.Examples of suitable 3′ regions are (1) the 3′ transcribed,non-translated regions containing the polyadenylation signal ofAgrobacterium Ti plasmid genes, such as the nopaline synthase (NOS;Fraley et al., Proceedings of the National Academy of Sciences, USA(1983) 80: 4803-4807) gene, and (2) plant genes such as the soybeanstorage protein genes and the small subunit of theribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. An example of a3′ region is that from the ssRUBISCO E9 gene from pea (European PatentApplication 0385 962).

In an aspect, an expression cassette or sequence of interest providedherein may comprise a sequence encoding a cell factor that functions toincrease DNA repair, where the protein is selected from the groupconsisting of a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, adominant-negative Ku70, or any combination thereof.

In one aspect, an expression cassette provided herein comprises anucleic acid sequence that is not essentially homologous to anendogenous plant nucleic acid sequence. In another aspect, an expressioncassette provided herein comprises a nucleic acid sequence that is notessentially homologous to an endogenous plant gene. In another aspect,an expression cassette provided herein comprises a nucleic acid sequencethat is at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to an endogenous plant gene. In another aspect,an expression cassette provided herein comprises a nucleic acid sequencethat is at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to an endogenous plant nucleic acid sequence.

Promoters

A promoter contains a sequence of nucleotide bases that signals RNApolymerase to associate with the DNA and to initiate transcription intomRNA using one of the DNA strands as a template to make a correspondingcomplementary strand of RNA. In an aspect, a promotor provided herein isa constitutive promoter. In another aspect, a promoter provided hereinis a regulatable promoter. In an aspect, an expression cassette providedherein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10promoters. In an aspect, a promoter provided herein is located within asequence of interest. In another aspect, a promoter provided herein isnot located within a sequence of interest.

A number of promoters that are active in plant cells have been describedin the literature. Such promoters would include but are not limited tothe nopaline synthase (NOS) and octopine synthase (OCS) promoters thatare carried on Ti plasmids of Agrobacterium tumefaciens, thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Sand 35S promoters and the Figwort mosaic virus (FMV) 35S promoter, andthe enhanced CaMV35S promoter (e35S). A variety of other plant genepromoters that are regulated in response to environmental, hormonal,chemical, and/or developmental signals, also can be used for expressionof heterologous genes in plant cells, including, for instance, promotersregulated by (1) heat (Callis et al., Plant Physiology, (1988) 88:965-968), (2) light (e.g., pea RbcS-3A promoter, Kuhlemeier et al.,Plant Cell, (1989) 1: 471-478; maize RbcS promoter, Schaffner et al.,Plant Cell (1991) 3: 997-1012); (3) hormones, such as abscisic acid(Marcotte et al., Plant Cell, (1989) 1: 969-976), (4) wounding (e.g.,Siebertz et al., Plant Cell, (1989) 961-968); or other signals orchemicals. Tissue specific promoters are also known.

In some embodiments, a promoter is capable of causing sufficientexpression to result in the production of an effective amount of thegene product of interest. Examples describing such promoters includewithout limitation U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S.Pat. No. 5,641,876 (rice actin promoter), U.S. Pat. No. 6,426,446 (maizeRS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S.Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611(constitutive maize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605,5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maizeL3 oleosin promoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter aswell as a rice actin 2 intron), U.S. Pat. No. 5,837,848 (root specificpromoter), U.S. Pat. No. 6,294,714 (light inducible promoters), U.S.Pat. No. 6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138(pathogen inducible promoters), U.S. Pat. No. 6,175,060 (phosphorusdeficiency inducible promoters), U.S. Pat. No. 6,635,806 (gamma-coixinpromoter), and U.S. patent application Ser. No. 09/757,089 (maizechloroplast aldolase promoter). Additional promoters that can find useare a nopaline synthase (NOS) promoter (Ebert et al., 1987), theoctopine synthase (OCS) promoter (which is carried on tumor-inducingplasmids of Agrobacterium tumefaciens), the caulimovirus promoters suchas the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al.,Plant Molecular Biology (1987) 9: 315-324), the CaMV 35S promoter (Odellet al., Nature (1985) 313: 810-812), the figwort mosaic virus35S-promoter (U.S. Pat. Nos. 6,051,753; 5,378,619), the sucrose synthasepromoter (Yang and Russell, Proceedings of the National Academy ofSciences, USA (1990) 87: 4144-4148), the R gene complex promoter(Chandler et al., Plant Cell (1989) 1: 1175-1183), and the chlorophylla/b binding protein gene promoter, PC1SV (U.S. Pat. No. 5,850,019), andAGRtu.nos (GenBank Accession V00087; Depicker et al., Journal ofMolecular and Applied Genetics (1982) 1: 561-573; Bevan et al., 1983)promoters.

In some embodiments, promoter hybrids can be constructed to enhancetranscriptional activity (U.S. Pat. No. 5,106,739), or to combinedesired transcriptional activity, inducibility and tissue specificity ordevelopmental specificity. Promoters that function in plants include butare not limited to promoters that are inducible, viral, synthetic,constitutive, temporally regulated, spatially regulated, andspatio-temporally regulated. Other promoters that are tissue-enhanced,tissue-specific, or developmentally regulated are also known in the artand envisioned to have utility in the practice of this disclosure.

Promoters used in the provided nucleic acid molecules and vectors ofthis disclosure can be modified, if desired, to affect their controlcharacteristics. Promoters can be derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters can be altered to contain multiple “enhancer sequences” toassist in elevating gene expression.

Selectable Markers

In one aspect, a sequence of interest provided herein can comprise oneor more selectable or screenable marker genes. In some embodiments theselectable or screenable marker gene aids in the identification of atransformed plant, or a product of agronomic utility. In someembodiments, DNA that serves as a selectable or screenable marker canfunction in a regenerable plant tissue to produce a compound that wouldconfer upon the plant tissue resistance to an otherwise toxic compound.A number of selectable or screenable marker genes are known in the artand can be used. Genes for use as a selectable or screenable marker caninclude, but are not limited, to β-glucuronidase (GUS), greenfluorescent protein (GFP), luciferase (LUC), genes conferring toleranceto antibiotics like kanamycin (Dekeyser et al., Plant Physiology (1989)90: 217-223) or spectinomycin (e.g. spectinomycin aminoglycosideadenyltransferase (aadA); U.S. Pat. No. 5,217,902), genes that encodeenzymes that give tolerance to herbicides like glyphosate (e.g.5-enolpyruvylshikimate-3-phosphate synthase (EPSPS): Della-Cioppa etal., Bio/Technology (1987) 5: 579-584); U.S. Pat. Nos. 5,627,061;5,633,435; 6,040,497; 5,094,945; WO04074443, and WO04009761; glyphosateoxidoreductase (GOX; U.S. Pat. No. 5,463,175); glyphosate decarboxylase(WO05003362 and US Patent Application 20040177399; or glyphosateN-acetyltransferase (GAT): Castle et al., Science (2004) 304: 1151-1154)U.S. Patent Publication 20030083480), dalapon (e.g. dehI encoding2,2-dichloropropionic acid dehalogenase conferring tolerance to2,2-dichloropropionic acid (Dalapon; WO9927116)), bromoxynil(haloarylnitrilase (Bxn) for conferring tolerance to bromoxynil(WO8704181A1; U.S. Pat. No. 4,810,648; WO8900193A), sulfonyl herbicides(e.g. acetohydroxyacid synthase or acetolactate synthase conferringtolerance to acetolactate synthase inhibitors such as sulfonylurea,imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide;(U.S. Pat. Nos. 6,225,105; 5,767,366; 4,761,373; 5,633,437; 6,613,963;5,013,659; 5,141,870; 5,378,824; 5,605,011); encoding ALS, GST-II),bialaphos or phosphinothricin or derivatives (e.g. phosphinothricinacetyltransferase (bar) conferring tolerance to phosphinothricin orglufosinate (U.S. Pat. Nos. 5,646,024, 5,561,236, 5,276,268; 5,637,489;5,273,894; and EP 275,957), atrazine (encoding GST-III), dicamba(dicamba monooxygenase; U.S. Patent Application Publications20030115626, 20030135879), or sethoxydim (modified acetyl-coenzyme Acarboxylase for conferring tolerance to cyclohexanedione (sethoxydim)and aryloxyphenoxypropionate (haloxyfop) (U.S. Pat. No. 6,414,222),among others. Other selection procedures can also be implementedincluding positive selection mechanisms (e.g. use of the manA gene ofEscherichia coli, allowing growth in the presence of mannose), and dualselection (e.g. simultaneously using 75-100 ppm spectinomycin and 3-10ppm glufosinate, or 75 ppm spectinomycin and 0.2-0.25 ppm dicamba) andwould still fall within the scope of this disclosure. Use ofspectinomycin at a concentration of about 25-1000 ppm, such as at about150 ppm, is also contemplated.

In one aspect, a selectable or screenable marker provided herein is apositive selection marker. A positive selection marker confers anadvantage to a cell comprising such marker. In an aspect, a sequence ofinterest provided herein comprises one or more positive selectionmarkers. In some embodiments, a positive selection marker confersantibiotic resistance or herbicide resistance. In another aspect, aselectable or screenable marker provided herein is a negative selectionmarker. In some embodiments, a sequence of interest provided hereincomprises one or more negative selection markers. In another aspect, aselectable or screenable marker provided herein is both a positiveselection marker and a negative selection marker. A negative selectablemarker provided herein can be a lethal or non-lethal negative selectablemarker. Examples of non-lethal negative selectable markers include U.S.Publication No. 2004-0237142, such as GGPP synthases, GA 2-oxidase genesequences, isopentenyltransferase (IPT), CKI1 (cytokinin-independent 1),ESR-2, ESR1-A, auxin-producing genes, such as indole-3-acetic acid(IAA), iaaM, iaah, roLABC, genes that result in over-expression ofethylene biosynthetic enzymes, VP1 genes, AB13 genes, LEC1 genes, andBas1 genes for example. A non-lethal negative selectable marker gene canbe included on any nucleic acid molecule provided herein. A non-lethalnegative selectable marker gene provided herein is a gene resulting inthe over-expression of a class of enzymes that use substrates of thegibberellic acid (GA) biosynthetic pathway, but that do not result inthe production of bioactive GA. In another aspect, a nucleic acidmolecule provided herein comprises a non-lethal negative selectablemarker gene such as a phytoene synthase gene from Erwinia herbicola(crtB).

In one aspect, by employing a selectable or screenable marker, one canprovide or enhance the ability to identify transformants. In someembodiments, the selectable or screenable marker imparts a distinctphenotype to cells expressing the marker protein and often provide ameans to more efficiently distinguish such transformed cells from cellsthat do not have the selectable or screenable marker. Such genes mayencode either a selectable or screenable marker, depending on whetherthe marker confers a trait which one can “select” for by chemical means,such as through the use of a selective agent (for example, an herbicide,or an antibiotic), or whether it is simply a trait that one can identifythrough observation (for example, expression of GFP) or testing or“screening”. Of course, many examples of suitable marker proteins areknown to the art and can be employed in the practice of the invention.

Included within the terms “selectable” or “screenable markers” also aregenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which are secretable antigens that can be identified byantibody interaction, or even secretable enzymes which can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, by ELISA; orsmall active enzymes detectable in extracellular solution (for example,α-amylase, β-lactamase, phosphinothricin acetyltransferase); andproteins that are inserted or trapped in the cell wall (for example,proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S).

Many selectable marker coding regions are known and could be usedincluding, but not limited to, neo (Potrykus et al., 1985), whichprovides kanamycin resistance and can be selected for using kanamycin,G418, paromomycin, etc.; bar, which confers bialaphos orphosphinothricin resistance; a mutant EPSP synthase protein (Hinchee etal., 1988) conferring glyphosate resistance; a nitrilase such as bxnfrom Klebsiella ozaenae which confers resistance to bromoxynil (Stalkeret al., 1988); a mutant acetolactate synthase (ALS) which confersresistance to imidazolinone, sulfonylurea, or other ALS inhibitingchemicals (European Patent Application 154,204, 1985); a methotrexateresistant DHFR (Thillet et al., 1988), a dalapon dehalogenase thatconfers resistance to the herbicide dalapon; or a mutated anthranilatesynthase that confers resistance to 5-methyl tryptophan.

Examples of screenable markers that may be employed include a$-glucuronidase (GUS) or uidA gene which encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., 1988); a β-lactamase gene(Sutcliffe, 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky et al., 1983) which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an α-amylase gene (Ikuta et al.,1990); a tyrosinase gene (Katz et al., 1983) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to form the easily-detectable compound melanin; aβ-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), whichallows for bioluminescence detection; an aequorin gene (Prasher et al.,1985) which may be employed in calcium-sensitive bioluminescencedetection; or a gene encoding for green fluorescent protein (Sheen etal., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tian et al.,1997; WO 97/41228).

Site-Specific Enzymes

In an aspect, a sequence of interest provided herein comprisespolynucleotides encoding at least 1, at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10 site-specific enzymes. In another aspect, a plant cell providedherein already comprises a polynucleotide encoding a site-specificenzyme. In an aspect, a polynucleotide encoding a site-specific enzymeprovided herein is stably transformed into a plant cell. In anotheraspect, a polynucleotide encoding a site-specific enzyme provided hereinis transiently transformed into a plant cell. In another aspect, apolynucleotide encoding a site-specific enzyme is under the control of aregulatable promoter, a constitutive promoter, a tissue specificpromoter, or any promoter useful for expression of the site-specificenzyme.

In one aspect, a vector comprises in cis a cassette encoding asite-specific enzyme and a sequence of interest such that when contactedwith the genome of a plant cell, the site-specific enzyme enablessite-specific integration of the sequence of interest. In one aspect, afirst vector comprises a cassette encoding a site-specific enzyme and asecond vector comprises a sequence of interest such that when contactedwith the genome of a plant cell, the site-specific enzyme provided intrans enables site-specific integration of the sequence of interest.

As used herein, the term “site-specific enzyme” refers to any enzymethat can cleave a nucleotide sequence in a site-specific manner. In anaspect, a site-specific enzyme provided herein is selected from thegroup consisting of an endonuclease (without being limiting, forexample, a meganuclease, a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nucleases (TALEN), an Argonaute (non-limitingexamples of Argonaute proteins include Thermus thermophilus Argonaute(TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacteriumgregoryi Argonaute (NgAgo), an RNA-guided nuclease (non-limitingexamples of RNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4,Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10,Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4,Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,Cpf1, CasX, CasY, homologs thereof, or modified versions thereof); arecombinase (without being limiting, for example, a serine recombinaseattached to a DNA recognition motif, a tyrosine recombinase attached toa DNA recognition motif); a transposase (without being limiting, forexample, a DNA transposase attached to a DNA binding domain); or anycombination thereof.

In an aspect, a tyrosine recombinase attached to a DNA recognition motifprovided herein is selected from the group consisting of a Crerecombinase, a Gin recombinase a Flp recombinase, and a Tnp1recombinase. In an aspect, a Cre recombinase or a Gin recombinaseprovided herein is tethered to a zinc-finger DNA binding domain. Inanother aspect, a serine recombinase attached to a DNA recognition motifprovided herein is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In another aspect, aDNA transposase attached to a DNA binding domain provided herein isselected from the group consisting of a TALE-piggyBac and TALE-Mutator.

Site-specific nucleases, such as meganucleases, ZFNs, TALENs, Argonauteproteins (non-limiting examples of Argonaute proteins include Thermusthermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo),Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, ormodified versions thereof), Cas9 nucleases (non-limiting examples ofRNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6,Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2,Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6,Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10,Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX,CasY, homologs thereof, or modified versions thereof), induce adouble-strand DNA break at the target site of a genomic sequence that isthen repaired by the natural processes of HR or NHEJ. Sequencemodifications then occur at the cleaved sites, which can includedeletions or insertions that result in gene disruption in the case ofNHEJ, or integration of nucleic acid sequences by HR.

In an aspect, a vector or sequence of interest provided herein cancomprise a nucleic acid sequence encoding a zinc finger nuclease. ZFNsare synthetic proteins consisting of an engineered zinc fingerDNA-binding domain fused to the cleavage domain of the FokI restrictionendonuclease. ZFNs can be designed to cleave almost any long stretch ofdouble-stranded DNA for modification of the zinc finger DNA-bindingdomain. ZFNs form dimers from monomers composed of a non-specific DNAcleavage domain of FokI endonuclease fused to a zinc finger arrayengineered to bind a target DNA sequence.

The DNA-binding domain of a ZFN is typically composed of 3-4 zinc-fingerarrays. The amino acids at positions −1, +2, +3, and +6 relative to thestart of the zinc finger o-helix, which contribute to site-specificbinding to the target DNA, can be changed and customized to fit specifictarget sequences. The other amino acids form the consensus backbone togenerate ZFNs with different sequence specificities. Rules for selectingtarget sequences for ZFNs are known in the art.

The FokI nuclease domain requires dimerization to cleave DNA andtherefore two ZFNs with their C-terminal regions are needed to bindopposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFNmonomer can cut the target site if the two-ZF-binding sites arepalindromic. The term ZFN, as used herein, is broad and includes amonomeric ZFN that can cleave double stranded DNA without assistancefrom another ZFN. The term ZFN is also used to refer to one or bothmembers of a pair of ZFNs that are engineered to work together to cleaveDNA at the same site.

Because the DNA-binding specificities of zinc finger domains can inprinciple be re-engineered using one of various methods, customized ZFNscan theoretically be constructed to target nearly any gene sequence.Publicly available methods for engineering zinc finger domains includeContext-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN),and Modular Assembly.

In an aspect, a vector or sequence of interest provided herein cancomprise a nucleic acid sequence encoding a transcription activator-likeeffector nuclease (TALEN). TALENs are artificial restriction enzymesgenerated by fusing the transcription activator-like effector (TALE) DNAbinding domain to a nuclease domain. In some embodiments, the nucleaseis selected from a group consisting of PvuII, MutH, TevI and FokI, AlwI,MIyI, Sbfl, SdaI, StsI, CleDORF, Clo051, Pept071. When each member of aTALEN pair binds to the DNA sites flanking a target site, the FokImonomers dimerize and cause a double-stranded DNA break at the targetsite.

The term TALEN, as used herein, is broad and includes a monomeric TALENthat can cleave double stranded DNA without assistance from anotherTALEN. The term TALEN is also used to refer to one or both members of apair of TALENs that work together to cleave DNA at the same site.

Transcription activator-like effectors (TALEs) can be engineered to bindpractically any DNA sequence. TALE proteins are DNA-binding domainsderived from various plant bacterial pathogens of the genus Xanthomonas.The X pathogens secrete TALEs into the host plant cell during infection.The TALE moves to the nucleus, where it recognizes and binds to aspecific DNA sequence in the promoter region of a specific DNA sequencein the promoter region of a specific gene in the host genome. TALE has acentral DNA-binding domain composed of 13-28 repeat monomers of 33-34amino acids. The amino acids of each monomer are highly conserved,except for hypervariable amino acid residues at positions 12 and 13. Thetwo variable amino acids are called repeat-variable diresidues (RVDs).The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognizeadenine, thymine, cytosine, and guanine/adenine, respectively, andmodulation of RVDs can recognize consecutive DNA bases. This simplerelationship between amino acid sequence and DNA recognition has allowedfor the engineering of specific DNA binding domains by selecting acombination of repeat segments containing the appropriate RVDs.

Besides the wild-type FokI cleavage domain, variants of the FokIcleavage domain with mutations have been designed to improve cleavagespecificity and cleavage activity. The FokI domain functions as a dimer,requiring two constructs with unique DNA binding domains for sites inthe target genome with proper orientation and spacing. Both the numberof amino acid residues between the TALEN DNA binding domain and the FokIcleavage domain and the number of bases between the two individual TALENbinding sites are parameters for achieving high levels of activity.PvuII, MutH, and TevI cleavage domains are useful alternatives to FokIand FokI variants for use with TALEs. PvuII functions as a highlyspecific cleavage domain when coupled to a TALE (see Yank et al. 2013.PloS One. 8: e82539). MutH is capable of introducing strand-specificnicks in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41:e83). TevI introduces double-stranded breaks in DNA at targeted sites(see Beurdeley et al., 2013. Nature Communications. 4: 1762).

The relationship between amino acid sequence and DNA recognition of theTALE binding domain allows for designable proteins. Software programssuch as DNA Works can be used to design TALE constructs. Other methodsof designing TALE constructs are known to those of skill in the art. SeeDoyle et al., Nucleic Acids Research (2012) 40: W117-122.; Cermak etal., Nucleic Acids Research (2011). 39:e82; andtale-nt.cac.comell.edu/about.

In an aspect, a vector or sequence of interest provided herein cancomprise a nucleic acid sequence encoding a meganuclease. Meganucleases,which are commonly identified in microbes, are unique enzymes with highactivity and long recognition sequences (>14 bp) resulting insite-specific digestion of target DNA. Engineered versions of naturallyoccurring meganucleases typically have extended DNA recognitionsequences (for example, 14 to 40 bp).

The engineering of meganucleases is more challenging than that of ZFNsand TALENs because the DNA recognition and cleavage functions ofmeganucleases are intertwined in a single domain. Specialized methods ofmutagenesis and high-throughput screening have been used to create novelmeganuclease variants that recognize unique sequences and possessimproved nuclease activity.

In an aspect, a vector provided herein can comprise any combination of anucleic acid sequence encoding an Argonaute (non-limiting examples ofArgonaute proteins include Thermus thermophilus Argonaute (TtAgo),Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryiArgonaute (NgAgo), homologs thereof, or modified versions thereof), andoptionally, a sequence encoding DNA guide.

In an aspect, a vector provided herein can comprise any combination of anucleic acid sequence encoding a RNA-guided Cas9 nuclease (non-limitingexamples of RNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4,Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10,Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4,Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,Cpf1, CasX, CasY, homologs thereof, or modified versions thereof); and,optionally, the guide RNA necessary for targeting the respectivenucleases.

Cas9 nucleases are part of the adaptive immune system of bacteria andarchaea, protecting them against invading nucleic acids such as virusesby cleaving the foreign DNA in a sequence-dependent manner. The immunityis acquired by the integration of short fragments of the invading DNAknown as spacers between two adjacent repeats at the proximal end of aCRISPR locus. The CRISPR arrays, including the spacers, are transcribedduring subsequent encounters with invasive DNA and are processed intosmall interfering CRISPR RNAs (crRNAs) approximately 40 nt in length,which combine with the trans-activating CRISPR RNA (tracrRNA) toactivate and guide the Cas9 nuclease. This cleaves homologousdouble-stranded DNA sequences known as protospacers in the invading DNA.A prerequisite for cleavage is the presence of a conservedprotospacer-adjacent motif (PAM) downstream of the target DNA, whichusually has the sequence 5-NGG-3 but less frequently NAG. Specificity isprovided by the so-called “seed sequence” approximately 12 basesupstream of the PAM, which must match between the RNA and target DNA.Cpf1 acts in a similar manner to Cas9, but Cpf1 does not require atracrRNA.

Site-Specific Enzyme Target Sites

In an aspect, a nucleic acid molecule provided herein comprises at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 site-specific enzyme targetsites. In an aspect, a vector or sequence of interest provided hereincomprises a Cre/lox recombination site, a Flp/FRT recombination site, anendonuclease recognition site, a TALEN site, or any combination thereof.In another aspect, a vector or sequence of interest provided hereincomprises a Cre recombinase or a Flp recombination system. In an aspect,a recombination system provided herein can act in cis. In anotheraspect, a recombination system provided herein can act in trans.

In an aspect, a site-specific enzyme target site provided herein is atleast 10, at least 20, at least 30, at least 40, at least 50, at least75, at least 100, at least 125, at least 150, at least 200, at least250, at least 300, at least 400, or at least 500 nucleotides.

The Flp-FRT site-directed recombination system comes from the 2p plasmidfrom the baker's yeast Saccharomyces cerevisiae. In this system, Pprecombinase (flippase) recombines sequences between flippase recognitiontarget (FRT) sites. FRT sites comprise 34 nucleotides. Flp binds to the“arms” of the FRT sites (one arm is in reverse orientation) and cleavesthe FRT site at either end of an intervening nucleic acid sequence.After cleavage, Flp recombines nucleic acid sequences between two FRTsites.

Cre-lox is a site-directed recombination system derived from thebacteriophage P1 that is similar to the Flp-FRT recombination system.Cre-lox can be used to invert a nucleic acid sequence, delete a nucleicacid sequence, or translocate a nucleic acid sequence. In this system,Cre recombinase recombines a pair of lox nucleic acid sequences. Loxsites comprise 34 nucleotides, with the first and last 13 nucleotides(arms) being palindromic. During recombination, Cre recombinase proteinbinds to two lox sites on different nucleic acids and cleaves at the loxsites. The cleaved nucleic acids are spliced together (reciprocallytranslocated) and recombination is complete. In another aspect, a loxsite provided herein is a loxP, lox 2272, loxN, lox 511, lox 5171,lox71, lox66, M2, M3, M7, or M11 site.

Methods and Compositions for Use of Gene Modification in Plants

In one aspect, the instant disclosure provides a Rhizobiales cellcomprising at least one vector that is capable of forming twoessentially complementary T-strands. In another aspect, the instantdisclosure provides an Agrobacterium cell comprising at least one vectorthat is capable of forming two essentially complementary T-strands.

In one aspect, the instant disclosure provides a method of increasingthe rate of site directed integration of a sequence of interest,comprising contacting a plant cell with a Rhizobiales cell capable oftransforming the plant cell, where the Rhizobiales cell comprises atleast one vector. In another aspect, the instant disclosure provides amethod of transforming a plant cell, comprising contacting the plantcell with a Rhizobiales cell capable of transforming the plant cell,where the Rhizobiales cell comprises at least one vector capable offorming two T-strands that are essentially complementary in at least aportion of a of the T-strands.

In one embodiment, a vector disclosed herein comprises a first rightborder DNA sequence (RB1), a second right border DNA sequence (RB2), andat least one sequence of interest, where the RB1 is positioned in thevector to initiate synthesis of a first T-strand such that the sequenceof interest is in the sense orientation from the 5′ to 3′ end of thefirst T-strand; and the RB2 is positioned in the vector to initiatesynthesis of a second T-strand such that the sequence of interest is inthe anti-sense orientation from the 5′ to 3′ end of the second T-strand,and where the two T-strands resulting from initiation at RB1 and RB2 areessentially complementary to each other.

In another embodiment, a vector disclosed herein comprises a first rightborder DNA sequence (RB1), a second right border DNA sequence (RB2), atleast one sequence of interest, a first left border DNA sequence (LB1),and a second left border DNA sequence (LB2), where the vector isconfigured such that the RB1 is paired with the LB1, which arepositioned in the vector to initiate (RB1) and terminate (LB1) synthesisof a first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the RB2 ispaired with the LB2, which are positioned in the vector to initiate(RB2) and terminate (LB2) synthesis of a second T-strand such that thesequence of interest is in an anti-sense orientation relative to thesequence of interest in the first T-strand, and where the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryin at least a portion of the sequence of interest.

In another embodiment, a vector disclosed herein comprises a first rightborder DNA sequence (RB1), a second right border DNA sequence (RB2), atleast one sequence of interest, and a left border DNA sequence (LB),where the vector is configured such that the RB1 is paired with the LB,which are positioned in the vector to initiate (RB1) and terminate (LB)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andthe RB2 initiates synthesis of a second T-strand such that the sequenceof interest is in an anti-sense orientation relative to the sequence ofinterest in the first T-strand, and where the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the sequence of interest.

In another embodiment, a vector disclosed herein comprises a first rightborder DNA sequence (RB1), a second right border DNA sequence (RB2), atleast one sequence of interest, and a left border DNA sequence (LB),where the vector is configured such that the RB1 initiates synthesis ofa first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the RB2 ispaired with the LB, which are positioned in the vector to initiate (RB2)and terminate (LB) synthesis of a second T-strand such that the sequenceof interest is in an anti-sense orientation relative to the sequence ofinterest in the first T-strand, and where the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the sequence of interest.

In another embodiment, a vector disclosed herein comprises a firstsequence of interest and a second sequence of interest, where the firstsequence of interest is essentially identical to the second sequence ofinterest; and the vector further comprises a first right border DNAsequence (RB1) with a first left border DNA sequence (LB1) which arepositioned in the vector to initiate (RB1) and terminate (LB1) synthesisof a first T-strand such that the first sequence of interest is in thesense orientation from the 5′ to 3′ end of the first T-strand; and thevector further comprises a second right border DNA sequence (RB2) and asecond left border DNA sequence (LB2) which are positioned in the vectorto initiate (RB2) and terminate (LB2) synthesis of a second T-strandsuch that the second sequence of interest is in an anti-senseorientation relative to the first sequence of interest in the firstT-strand, and where the two T-strands resulting from initiation at RB1and RB2 are essentially complementary in at least a portion of the firstsequence of interest and the second sequence of interest.

In another embodiment, a vector disclosed herein comprises a firstsequence of interest and a second sequence of interest, where the firstsequence of interest is essentially identical to the second sequence ofinterest; and the vector further comprises a first right border DNAsequence (RB1) with a left border DNA sequence (LB) which are positionedin the vector to initiate (RB1) and terminate (LB) synthesis of a firstT-strand such that the first sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the vectorfurther comprises a second right border DNA sequence (RB2) whichinitiates synthesis of a second T-strand such that the second sequenceof interest is in an anti-sense orientation relative to the firstsequence of interest in the first T-strand, and where the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryin at least a portion of the first sequence of interest and the secondsequence of interest.

In another embodiment, a vector disclosed herein comprises a firstsequence of interest and a second sequence of interest, where the firstsequence of interest is essentially identical to the second sequence ofinterest; and the vector further comprises a first right border DNAsequence (RB1) which initiates synthesis of a first T-strand such thatthe first sequence of interest is in the sense orientation from the 5′to 3′ end of the first T-strand; and the vector further comprises asecond right border DNA sequence (RB2) and a left border DNA sequence(LB) which are positioned in the vector to initiate (RB2) and terminate(LB) synthesis of a second T-strand such that the second sequence ofinterest is in an anti-sense orientation relative to the first sequenceof interest in the first T-strand, and where the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary in at leasta portion of the first sequence of interest and the second sequence ofinterest.

In another embodiment, a vector disclosed herein comprises a firstsequence of interest and a second sequence of interest, where the firstsequence of interest is essentially identical to the second sequence ofinterest; and the vector further comprises a first right border DNAsequence (RB1) to initiate synthesis of a first T-strand such that thefirst sequence of interest is in the sense orientation from the 5′ to 3′end of the first T-strand; and the vector further comprises a secondright border DNA sequence (RB2) which initiates synthesis of a secondT-strand such that the second sequence of interest is in an anti-senseorientation relative to the first sequence of interest in the firstT-strand, and where the two T-strands resulting from initiation at RB1and RB2 are essentially complementary in at least a portion of the firstsequence of interest and the second sequence of interest.

In yet another embodiment, a Rhizobiales cell disclosed herein comprisesat least a first vector and a second vector, where each vector comprisesessentially identical sequences of interest, and where the first vectorcomprises a first right border DNA sequence (RB1) and a first leftborder DNA sequence (LB1) which are positioned in the first vector toinitiate (RB1) and terminate (LB1) synthesis of a first T-strand suchthat the sequence of interest is in the sense orientation from the 5′ to3′ end of the first T-strand; and the second vector comprises a secondright border DNA sequence (RB2) and a second left border DNA Sequence(LB2) which are positioned in the second vector to initiate (RB2) andterminate (LB2) synthesis of a second T-strand such that the sequence ofinterest is in an anti-sense orientation relative to the sequence ofinterest in the first T-strand, and where the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary to eachother in at least a portion of the sequence of interest.

In another embodiment, a Rhizobiales cell provided herein comprises atleast a first vector and a second vector, where each vector comprisesessentially identical sequences of interest, and where the first vectorcomprises a first right border DNA sequence (RB1) and a left border DNAsequence (LB) which are positioned in the first vector to initiate (RB1)and terminate (LB) synthesis of a first T-strand such that the sequenceof interest is in the sense orientation from the 5′ to 3′ end of thefirst T-strand; and the second vector comprises a second right borderDNA sequence (RB2) which initiates synthesis of a second T-strand suchthat the sequence of interest is in an anti-sense orientation relativeto the sequence of interest in the first T-strand, and where the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other in at least a portion of the sequence ofinterest.

In another embodiment, a Rhizobiales cell provided herein comprises atleast a first vector and a second vector, where each vector comprisesessentially identical sequences of interest, and where the first vectorcomprises a first right border DNA sequence (RB1) which initiatessynthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andthe second vector comprises a second right border DNA sequence (RB2) anda left border DNA Sequence (LB) which are positioned in the secondvector to initiate (RB2) and terminate (LB) synthesis of a secondT-strand such that the sequence of interest is in an anti-senseorientation relative to the sequence of interest in the first T-strand,and where the two T-strands resulting from initiation at RB1 and RB2 areessentially complementary to each other in at least a portion of thesequence of interest.

In another embodiment, a Rhizobiales cell provided herein comprises atleast a first vector and a second vector, where each vector comprisesessentially identical sequences of interest, and where the first vectorcomprises a first right border DNA sequence (RB1) which initiatessynthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andthe second vector comprises a second right border DNA sequence (RB2)which initiates synthesis of a second T-strand such that the sequence ofinterest is in an anti-sense orientation relative to the sequence ofinterest in the first T-strand, and where the two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary to eachother in at least a portion of the sequence of interest.

In one aspect, methods provided in the instant disclosure comprise twoor more vectors, where the two or more vectors are in one Rhizobialescell. In another aspect, methods provided in the instant disclosurecomprise two or more vectors, where the two or more vectors are in twoor more Rhizobiales cells. For example, a first Rhizobiales cellcomprises a first vector, and a second Rhizobiales cell comprises asecond vector.

In one aspect, the instant disclosure provides a method of increasingthe rate of site directed integration of a sequence of interest,comprising contacting the plant cell with two or more Rhizobiales cellscapable of transforming the plant cell, where the two or moreRhizobiales cells each contain one of at least two vectors capable offorming two essentially complementary T-strands.

In another aspect, the instant disclosure provides a method oftransforming a plant cell, comprising contacting the plant cell with twoor more Rhizobiales cells capable of transforming the plant cell, wherethe two or more Rhizobiales cells each contain one of at least twovectors capable of forming two essentially complementary T-strands. Inanother aspect, the instant disclosure provides a method of increasingthe rate of site directed integration of a sequence of interest,comprising contacting a plant cell with two or more Rhizobiales cells,where the two or more Rhizobiales cells each contain one of at least twovectors capable of forming two essentially complementary T-strands.

In one embodiment, a first Rhizobiales cell and a second Rhizobialescell provided herein contain at least a first vector and a secondvector, respectively, where each vector comprises an essentiallyidentical sequence of interest, and where the first vector comprises afirst right border DNA sequence (RB1), and where the RB1 is positionedin the vector to initiate synthesis of a first T-strand such that thesequence of interest is in the sense orientation from the 5′ to 3′ endof the first T-strand; and the second vector comprises a second rightborder DNA sequence (RB2) which is positioned in the vector to initiatesynthesis of a second T-strand such that the sequence of interest is inthe anti-sense orientation relative to the sequence of interest in thefirst T-strand, and where the two T-strands resulting from initiation atRB1 and RB2 are essentially complementary in at least a portion of thesequence of interest.

In another embodiment, a first Rhizobiales cell and a second Rhizobialescell provided herein contain at least a first vector and a secondvector, respectively, where each vector comprises an essentiallyidentical sequence of interest, and where the first vector comprises afirst right border DNA sequence (RB1) and a first left border DNAsequence (LB1) which are positioned in the first vector to initiate(RB1) and terminate (LB1) synthesis of a first T-strand such that thesequence of interest is in the sense orientation from the 5′ to 3′ endof the first T-strand; and the second vector comprises a second rightborder DNA sequence (RB2) and a second left border DNA sequence (LB2)which are positioned in the second vector to initiate (RB2) andterminate (LB2) synthesis of a second T-strand such that the sequence ofinterest is in an anti-sense orientation from the 5′ to 3′ end of thesecond T-strand, and where the sequence of interest in the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryto each other.

In one embodiment, the instant disclosure provides a method comprising afirst Rhizobiales cell and a second Rhizobiales cell, where eachRhizobiales cell contains at least one of two vectors, where each vectorcomprises an essentially identical sequence of interest, and where thefirst vector comprises a first right border DNA sequence (RB1), andwhere the RB1 is positioned in the vector to initiate synthesis of afirst T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the secondvector comprises a second right border DNA sequence (RB2) which ispositioned in the vector to initiate synthesis of a second T-strand suchthat the sequence of interest is in the anti-sense orientation relativeto the sequence of interest in the first T-strand, and wherein the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest.

In another embodiment, the instant disclosure provides a methodcomprising a first Rhizobiales cell and a second Rhizobiales cell, whereeach Rhizobiales cell contains at least one of two vectors, where eachvector comprises an essentially identical sequence of interest, andwhere the first vector comprises a first right border DNA sequence (RB1)and a first left border DNA sequence (LB1) which are positioned in thefirst vector to initiate (RB1) and terminate (LB1) synthesis of a firstT-strand such that the sequence of interest is in the sense orientationfrom the 5′ to 3′ end of the first T-strand; and the second vectorcomprises a second right border DNA sequence (RB2) and a second leftborder DNA sequence (LB2) which are positioned in the second vector toinitiate (RB2) and terminate (LB2) synthesis of a 30 second T-strandsuch that the sequence of interest is in an anti-sense orientation fromthe 5′ to 3′ end of the second T-strand, and wherein the sequence ofinterest in the two T-strands resulting from initiation at RB1 and RB2are essentially complementary to each other.

In another embodiment, the instant disclosure provides a methodcomprising a first Rhizobiales cell and a second Rhizobiales cell, whereeach Rhizobiales cell contains at least one of two vectors, where eachvector comprises an essentially identical sequence of interest, andwhere the first vector comprises a first right border DNA sequence (RB1)and a left border DNA sequence (LB) which are positioned in the firstvector to initiate (RB1) and terminate (LB) synthesis of a firstT-strand such that the sequence of interest is in the sense orientationfrom the 5′ to 3′ end of the first T-strand; and the second vectorcomprises a second right border DNA sequence (RB2) to initiate synthesisof a second T-strand such that the sequence of interest is in ananti-sense orientation from the 5′ to 3′ end of the second T-strand, andwherein the sequence of interest in the two T-strands resulting frominitiation at RB1 and RB2 are essentially complementary to each other.

In another embodiment, the instant disclosure provides a methodcomprising a first Rhizobiales cell and a second Rhizobiales cell, whereeach Rhizobiales cell contains at least one of two vectors, where eachvector comprises an essentially identical sequence of interest, andwhere the first vector comprises a first right border DNA sequence (RB1)initiate synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the second vector comprises a second right border DNAsequence (RB2) and a left border DNA sequence (LB) which are positionedin the second vector to initiate (RB2) and terminate (LB) synthesis of asecond T-strand such that the sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand, and whereinthe sequence of interest in the two T-strands resulting from initiationat RB1 and RB2 are essentially complementary to each other.

In another embodiment, at least one vector disclosed herein comprises anRB and a LB and where the vector further comprises between the RB andLB: (i) a first sequence of interest in a sense orientation relative tothe RB, (ii) a spacer, and (iii) a second sequence of interest in ananti-sense orientation relative to the RB, where the first sequence ofinterest and the second sequence of interest are essentiallycomplementary and after synthesis of the T-strand anneal to form adouble-stranded DNA.

In one aspect, the integration of at least part of the sequence ofinterest in a vector results in a point mutation, an insertion, adeletion, an inversion, increased transcription of an endogenous genomiclocus, decreased transcription of an endogenous genomic locus, alteredprotein activity, increased transcription of the sequence of interest,decreased transcription of the integrated sequence of interest, or acombination thereof.

In one aspect, the sequence of interest in the vector further comprisesat least one, at least two, at least three, at least four, at leavefive, at least six, at least seven, at least eight, at least nine, or atleast ten site-specific enzyme target sites. Examples of a site-specificenzyme target site include, but are not limited to, a Cre/loxrecombination site, a Flp/FRT recombination site, a endonucleaserecognition site, and a TALEN site.

In one aspect, the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., Sinorhizobium spp., and anEnsifer spp. In another aspect, the Rhizobiales cell is an Agrobacteriumcell. In yet another aspect, the Rhizobiales cell is an Agrobacteriumtumefaciens cell. In another aspect, the Rhizobiales cell furthercontains a vector comprising one or more gene expression cassettes withsequence encoding a cell factor that functions to increase DNA repair,one or more Agrobacterium Ti plasmid vir genes, one or more selectablemarker genes, an origin of replication, or any combination thereof.

Methods for Increasing Rates of Site-Directed Integration by ProlongedCo-Culturing

In an aspect, the instant disclosure provides a method of increasing therate of site-directed integration of a sequence of interest in a plantgenome, comprising contacting at least one plant cell on a co-culturemedium for at least two days, at least 3 days, at least 4 days or atleast 5 days with a Rhizobiales cell capable of transforming the plantcell, wherein the Rhizobiales cell comprises at least one vectordescribed in the instant disclosure.

In another aspect, the instant disclosure provides a method oftransforming a plant genome, comprising contacting at least one plantcell on a co-culture medium for at least two days, at least 3 days, atleast 4 days, or at least 5 days with at least one Rhizobiales cellcapable of transforming the plant cell, where the Rhizobiales cellcomprises at least one vector capable of forming two essentiallycomplementary T-strands.

In one aspect, the contacting comprises co-culturing the plant cell witha Rhizobiales cell for at least three days, at least four days, at leastfive days, at least six days, at least seven days, at least eight days,at least nine days, or at least ten days. In one aspect, the contactingcomprises co-culturing the plant cell with a Rhizobiales cell for atleast 48 hours, at least 49 hours, at least 50 hours, at least 51 hours,at least 52 hours, at least 53 hours, at least 54 hours, at least 55hours, at least 56 hours, at least 57 hours, at least 58 hours, at least59 hours, at least 60 hours, at least 61 hours, at least 62 hours, atleast 63 hours, at least 64 hours, at least 65 hours, at least 66 hours,at least 67 hours, at least 68 hours, at least 69 hours, at least 70hours, at least 71 hours, at least 72 hours, at least 73 hours, at least74 hours, at least 75 hours, at least 76 hours, at least 77 hours, atleast 78 hours, at least 79 hours, at least 80 hours, at least 81 hours,at least 82 hours, at least 83 hours, at least 84 hours, at least 85hours, at least 86 hours, at least 87 hours, at least 88 hours, at least89 hours, at least 90 hours, at least 91 hours, at least 92 hours, atleast 93 hours, at least 94 hours, at least 95 hours, at least 96 hours,at least 97 hours, at least 98 hours, at least 99 hours, at least 100hours, at least 101 hours, at least 102 hours, at least 103 hours, atleast 104 hours or at least 105 hours.

In one aspect, a Rhizobiales cell is selected from the group consistingof an Agrobacterium spp. cell, a Bradyrhizobium spp. cell, aMesorhizobium spp. cell, an Ochrobactrum spp. cell, a Phyllobacteriumspp. cell, a Rhizobium spp. cell, and a Sinorhizobium spp. cell. In afurther aspect, an Agrobacterium spp. cell is selected from the groupconsisting of an Agrobacterium tumefaciens cell and an Agrobacteriumrhizogenes cell.

In one aspect, the method further comprises detecting the integration ofat least a fragment of the sequence of interest of the vector in the atleast one plant cell. In one aspect, the at least one fragment of thesequence of interest in the vector is integrated into the plant genomeby HR. In another aspect, the at least one fragment of the sequence ofinterest in the vector is integrated into the plant genome by NHEJ.

In one aspect, the method also comprises selecting the plant call basedon the presence of the at least one fragment of the sequence of interestin the vector integrated into the plant genome. In a further aspect, themethod further comprises regenerating a transgenic plant form theselected plant cell.

In one aspect, a method or system for site-specific modification of atarget nucleic acid sequence provided herein involves homologousrecombination. In another aspect, a method or system for site-specificmodification of a target nucleic acid sequence provided herein involvesnon-homologous end joining. In yet another aspect, a method or systemfor site-specific modification of a target nucleic acid sequenceprovided herein comprises non-homologous end joining that furthercomprises the introduction of an insertion and/or deletion of one ormore, two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, twenty ormore, fifty or more, or one hundred or more nucleotides into the targetnucleic acid sequence.

Transformation Methods

Methods of transforming plant cells are well known by persons ofordinary skill in the art. For instance, specific instructions fortransforming plant cells by microprojectile bombardment with particlescoated with recombinant DNA are found in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812(wheat); U.S. Pat. No. 6,002,070 (rice); U.S. Pat. No. 7,122,722(cotton); U.S. Pat. No. 6,051,756 (Brassica); U.S. Pat. No. 6,297,056(Brassica); US Patent Publication 20040123342 (sugarcane) andAgrobacterium-mediated transformation is described in U.S. Pat. No.5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No.5,591,616 (corn); U.S. Pat. No. 6,384,301 (soybean); U.S. Pat. No.5,750,871 (Brassica); U.S. Pat. No. 5,463,174 (Brassica) 5,188,958(Brassica), all of which are incorporated herein by reference. Methodsfor transforming other plants can be found in, for example, Compendiumof Transgenic Crop Plants (2009) Blackwell Publishing. Any appropriatemethod known to those skilled in the art can be used to transform aplant cell with any of the nucleic acid molecules provided herein.

In one aspect, a method provided herein stably transforms a plant cell.In another aspect, a method provided herein transiently transforms aplant cell. In an aspect, a method of transforming a plant cell providedherein comprises a biolistic transformation or a bacteria-mediatedtransformation. In an aspect, a method of transforming a plant cellprovided herein comprises bacteria-mediated transformation that furthercomprises contacting the plant cell with a Rhizobiales cell, where theRhizobiales cell is capable of transforming the plant cell.

Transformation methods to provide transgenic plant cells and transgenicplants containing stably integrated nucleic acid molecules providedherein are preferably practiced in tissue culture on media and in acontrolled environment. “Media” refers to the numerous nutrient mixturesthat are used to grow cells in vitro, that is, outside of the intactliving organism.

In one aspect, this disclosure provides plant cells that are notreproductive material and do not mediate the natural reproduction of theplant. In another aspect, this disclosure also provides plant cells thatare reproductive material and mediate the natural reproduction of theplant. In another aspect, this disclosure provides plant cells thatcannot maintain themselves via photosynthesis. In another aspect, thisdisclosure provides somatic plant cells. Somatic cells, contrary togermline cells, do not mediate plant reproduction.

Recipient cell targets for transformation include, but are not limitedto, a seed cell, a fruit cell, a leaf cell, a cotyledon cell, ahypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, aroot cell, a shoot cell, a stem cell, a pod cell, a flower cell, aninflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigmacell, a receptacle cell, a petal cell, a sepal cell, a pollen cell, ananther cell, a filament cell, an ovary cell, an ovule cell, a pericarpcell, a phloem cell, a bud cell, or a vascular tissue cell. In anotheraspect, this disclosure provides a plant chloroplast. In a furtheraspect, this disclosure provides an epidermal cell, a stomata cell, atrichome cell, a root hair cell, a storage root cell, or a tuber cell.In another aspect, this disclosure provides a protoplast. In anotheraspect, this disclosure provides a plant callus cell. Any cell fromwhich a fertile plant can be regenerated is contemplated as a usefulrecipient cell for practice of this disclosure. Callus can be initiatedfrom various tissue sources, including, but not limited to, immatureembryos or parts of embryos, seedling apical meristems, microspores, andthe like. Those cells which are capable of proliferating as callus canserve as recipient cells for transformation. Practical transformationmethods and materials for making transgenic plants of this disclosure(e.g., various media and recipient target cells, transformation ofimmature embryos, and subsequent regeneration of fertile transgenicplants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and6,232,526 and U. S. Patent Application Publication 2004/0216189.

In one aspect, the instant disclosure provides a plant cell transformedby any method provided herein. In an aspect, a plant cell providedherein is selected from the group consisting of an Acacia cell, analfalfa cell, an aneth cell, an apple cell, an apricot cell, anartichoke cell, an arugula cell, an asparagus cell, an avocado cell, abanana cell, a barley cell, a bean cell, a beet cell, a blackberry cell,a blueberry cell, a broccoli cell, a Brussels sprout cell, a cabbagecell, a canola cell, a cantaloupe cell, a carrot cell, a cassava cell, acauliflower cell, a celery cell, a Chinese cabbage cell, a cherry cell,a cilantro cell, a citrus cell, a clementine cell, a coffee cell, a corncell, a cotton cell, a cucumber cell, a Douglas fir cell, an eggplantcell, an endive cell, an escarole cell, an eucalyptus cell, a fennelcell, a fig cell, a forest tree cell, a gourd cell, a grape cell, agrapefruit cell, a honey dew cell, a jicama cell, kiwifruit cell, alettuce cell, a leek cell, a lemon cell, a lime cell, a Loblolly pinecell, a mango cell, a maple tree cell, a melon cell, a mushroom cell, anectarine cell, a nut cell, an oat cell, an okra cell, an onion cell, anorange cell, an ornamental plant cell, a papaya cell, a parsley cell, apea cell, a peach cell, a peanut cell, a pear cell, a pepper cell, apersimmon cell, a pine cell, a pineapple cell, a plantain cell, a plumcell, a pomegranate cell, a poplar cell, a potato cell, a pumpkin cell,a quince cell, a radiata pine cell, a radicchio cell, a radish cell, arapeseed cell, a raspberry cell, a rice cell, a rye cell, a sorghumcell, a Southern pine cell, a soybean cell, a spinach cell, a squashcell, a strawberry cell, a sugar beet cell, a sugarcane cell, asunflower cell, a sweet corn cell, a sweet potato cell, a sweetgum cell,a tangerine cell, a tea cell, a tobacco cell, a tomato cell, a turfcell, a vine cell, watermelon cell, a wheat cell, a yam cell, and azucchini cell. In another aspect, a plant cell provided herein isselected from the group consisting of a corn cell, a soybean cell, acanola cell, a cotton cell, a wheat cell, and a sugarcane cell.

In another aspect, a plant cell provided herein is selected from thegroup consisting of a corn immature embryo cell, a corn mature embryocell, a corn seed cell, a soybean immature embryo cell, a soybean matureembryo cell, a soybean seed cell, a canola immature embryo cell, acanola mature embryo cell, a canola seed cell, a cotton immature embryocell, a cotton mature embryo cell, a cotton seed cell, a wheat immatureembryo cell, a wheat mature embryo cell, a wheat seed cell, a sugarcaneimmature embryo cell, a sugarcane mature embryo cell, a sugarcane seedcell.

In one aspect, transformation of a plant cell is performed by anAgrobacterium or other Rhizobiales-mediated method (U.S. Pat. Nos.6,265,638, 5,731,179; U.S. Patent Application PublicationsUS2005/0183170; 2003110532). The polynucleotide sequences that can betransferred into a plant cell provided herein can be present on onerecombination vector in one bacterial strain being utilized fortransformation. In another aspect, the polynucleotide sequences providedherein can be present on separate recombination vectors in one bacterialstrain. In yet another aspect, the polynucleotide sequences providedherein can be found in separate bacterial cells or strains used togetherfor transformation.

The DNA constructs used for transformation in the methods of presentdisclosure generally also contain the plasmid backbone DNA segments thatprovide replication function and antibiotic selection in bacterialcells, for example, an Escherichia coli origin of replication such asori322, an Agrobacterium origin of replication such as oriV or oriRi,and a coding region for a selectable marker such as Spec/Strp thatencodes for Tn7 aminoglycoside adenyltransferase (aadA) conferringresistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent)selectable marker gene. For plant transformation, the host bacterialstrain is often Agrobacterium tumefaciens ABI, C58, LBA4404, AGLO, AGL1,EHA101, or EHA105 carrying a plasmid having a transfer function for theexpression unit. Other strains known to those skilled in the art ofplant transformation can function in this disclosure.

To confirm the presence of integrated DNA in a transformed cell orgenome a variety of assays can be performed. Such assays include, forexample, molecular biological assays (e.g., Southern and northernblotting, PCR™); biochemical assays, such as detecting the presence of aprotein product (e.g., by immunological means (ELISAs and westernblots), or by enzymatic function (e.g., GUS assay)); pollenhistochemistry; plant part assays, (e.g., leaf or root assays); andalso, by analyzing the phenotype of the whole regenerated plant.

The instant disclosure also provides a transgenic plant cell comprisinga sequence of interest integrated into a genome of the plant cellaccording to the methods disclosed herein. Also provided is a transgenicplant produced by the methods disclosed herein.

EXAMPLES Example 1. Construct of Control Vectors

Two control vectors were created as non-limiting examples forAgrobacterium-mediated transformation. Both control vectors contain asequence of interest comprising three expression cassettes: anexpression cassette encoding a gene (CP4-EPSPS) to confer tolerance tothe herbicide glyphosate positioned between a left homology arm and aright homology arm; and two expression cassettes each encoding half of aTALEN pair. TALENs were obtained from Life Technologies. The firstcontrol vector comprised a RB DNA sequence, the sequence of interestcomprising the three expression cassettes, and a LB DNA sequence, asillustrated in FIG. 4A. The second control vector was the same as thefirst control vector except that there was no LB DNA sequence. These twocontrol vectors were used in Example 9, below.

Example 2. Construct of a Vector with Two RB DNA Sequences

A vector comprising two RB DNA sequences, and zero LB DNA sequences, wascreated for Agrobacterium-mediated transformation (FIG. 4B). The vectorwas constructed to contain a sequence of interest comprising threeexpression cassettes: one expression cassette encoding a gene(CP4-EPSPS) to confer tolerance to the herbicide glyphosate positionedbetween a left homology arm and a right homology arm; and two expressioncassettes each encoding half of a TALEN pair; and two RB DNA sequences(RB1 and RB2). This vector was used in Example 9 below. The vectorconfiguration comprised the first RB DNA sequence (RB1) positioned inthe vector to initiate synthesis of a first T-strand comprising thethree expression cassettes (CP4-EPSPS and two TALENs); and the second RBDNA sequence (RB2) was positioned in the vector to initiate synthesis ofa second T-strand comprising the three expression cassettes (CP4-EPSPSand two TALENs) such that the second T-strand was in an anti-senseorientation relative to the first T-strand. The two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary to eachother in at least a portion of the sequence of interest.

Example 3. Construct of a Vector with Two RB DNA Sequences and One LBDNA Sequence

A vector comprising two RB DNA sequences, and one LB DNA sequence, wascreated for Agrobacterium-mediated transformation (FIG. 4C). The vectorwas constructed to contain a sequence of interest comprising threeexpression cassettes: one expression cassette encoding a gene(CP4-EPSPS) to confer tolerance to the herbicide glyphosate positionedbetween a left homology arm and a right homology arm; and two expressioncassettes each encoding half of a TALEN pair; and two right border DNAsequences (RB1 and RB2), and one LB DNA sequence (LB1) paired with thefirst RB DNA sequence (RB1). The vector configuration comprised thefirst RB DNA sequence (RB1) positioned in the vector to initiatesynthesis of a first T-strand comprising the three expression cassettes(CP4-EPSPS and two TALENs) and terminate synthesis at the first LB DNAsequence (LB1) (paired with RB1); and the second RB DNA sequence (RB2)was positioned in the vector to initiate synthesis of a second T-strandcomprising the three expression cassettes (CP4-EPSPS and two TALENs)such that the second T-strand was in an anti-sense orientation relativeto the first T-strand. This vector was used in Example 9 below. The twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other in at least a portion of the sequence ofinterest.

Example 4. Construct of a Vector with Two RB DNA Sequences and Two LBDNA Sequences

A vector comprising at least one sequence of interest, two RB DNAsequences, and two LB DNA sequences is created forAgrobacterium-mediated transformation (a non-limiting example ispresented in FIG. 1D). The vector configuration comprises a first RB DNAsequence (RB1) paired with a first LB DNA sequence (LB1) which arepositioned in the vector to initiate (RB1) and terminate (LB1) synthesisof a first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the secondRB DNA sequence (RB2) is paired with a second LB DNA sequence (LB2)which are positioned in the vector to initiate synthesis of a secondT-strand such that the sequence of interest is in an anti-senseorientation relative to the sequence of interest in the first T-strand.The two T-strands resulting from initiation at RB1 and RB2 areessentially complementary to each other in at least a portion of thesequence of interest.

Example 5. Construct of a Vector with Two RB DNA Sequences and TwoEssentially Identical Sequences of Interest

A vector comprising two essentially identical sequences of interest, atleast two RB DNA sequences, and optional one or more LB DNA sequences iscreated for Agrobacterium-mediated transformation (a non-limitingexample is presented in FIG. 2A). The vector configuration comprises afirst RB DNA sequence (RB1) with an optional first LB DNA sequence (LB1)which are positioned in the vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the first sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the vector configuration further comprises a second RB DNA sequence(RB2) and an optional second LB DNA sequence (LB2) which are positionedin the vector to initiate (RB2) and terminate (LB2) synthesis of asecond T-strand such that the second sequence of interest is in ananti-sense orientation relative to the sequence of interest in the firstT-strand. The two T-strands resulting from initiation at RB1 and RB2 areessentially complementary to each other in at least a portion of thesequence of interest.

Example 6. Construct of Two Vectors for Co-Transformation

Two vectors are provided for the co-transformation of plant cells withAgrobacterium-mediated transformation (a non-limiting example ispresented in FIG. 2B). The two vector configurations compriseessentially identical sequences of interest, where the first vectorconfiguration comprises a first RB DNA sequence (RB1) and an optionalfirst LB DNA sequence (LB1) which are positioned in the first vector toinitiate (RB1) and terminate (LB1) synthesis of a first T-strand suchthat the sequence of interest is in the sense orientation from the 5′ to3′ end of the first T-strand; and the second vector configurationcomprises a second RB DNA sequence (RB2) and an optional second LB DNAsequence (LB2) which are positioned in the second vector to initiate(RB2) and terminate (LB2) synthesis of a second T-strand such that thesequence of interest is in an anti-sense orientation relative to thesequence of interest in the first T-strand. The two T-strands resultingfrom initiation at RB1 and RB2 are essentially complementary to eachother in at least a portion of the sequence of interest. The two vectorsmay be in the same Agrobacterium cell, or the two vectors may be indifferent Agrobacterium cells.

Example 7. Construct of a Vector with Two Different Sequences ofInterest and Three or More RB DNA Sequences

A Vector Comprising a First Sequence of Interest, a Second Sequence ofInterest different from the first sequence of interest, three or more RBDNA sequences, and optional one or more LB DNA sequences is created forAgrobacterium-mediated transformation. For illustrative purposes, onenon-limiting example of a vector configuration is presented in FIG. 2Cwhere the vector configuration comprises a first RB DNA sequence (RB1)and a first LB DNA sequence (LB1) which are positioned in the vector toinitiate (RB1) and terminate (LB1) synthesis of a first T-strand suchthat the first sequence of interest is in the sense orientation from the5′ to 3′ end of the first T-strand; and the vector configuration furthercomprises a second RB DNA sequence (RB2) and a second LB DNA sequence(LB2) which are positioned in the vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the first sequence ofinterest is in an anti-sense orientation relative to the sequence ofinterest in the first T-strand. The two T-strands resulting frominitiation at RB1 and RB2 are essentially complementary to each other inat least a portion of the sequence of interest. The vector configurationfurther comprises a third RB DNA sequence (RB3) and a third LB DNAsequence (LB3) which are positioned in the vector to initiate (RB3) andterminate (LB3) synthesis of a third T-strand such that the secondsequence of interest is in the sense orientation from the 5′ to 3′ endof the third T-strand; and the vector configuration further comprises afourth RB DNA sequence (RB4) and a fourth LB DNA sequence (LB4) whichare positioned in the vector to initiate (RB4) and terminate (LB4)synthesis of a fourth T-strand such that the second sequence of interestis in an anti-sense orientation relative to the sequence of interest inthe third T-strand. The two T-strands resulting from initiation at RB3and RB4 are essentially complementary to each other in at least aportion of the sequence of interest.

Example 8. Construct of a Vector with a RB DNA Sequence, a LB DNASequence, and Two Sequences of Interest Linked by a Spacer

A vector is provided for Agrobacterium-mediated transformation where thevector configuration comprises a RB DNA sequence and a LB DNA sequenceand where the vector further comprises between the RB and LB DNAsequences: (i) a first sequence of interest in a sense orientationrelative to the RB DNA sequence, (ii) a spacer, and (iii) a secondsequence of interest in an anti-sense orientation relative to the firstsequence of interest. The two sequences of interest, after synthesis ofthe T-strand, form a partially double stranded hairpin structure due tocomplementary base pairing. For illustrative purposes, one non-limitingexample of such a vector configuration is presented in FIG. 3A.

Example 9. Increased Frequency of Site-Directed Integration byAgrobacterium-Mediated Transformation

A locus termed L7 was identified as occurring only once in a corngenome. A site was selected within the L7 locus for insertion of atransgene by site-directed recombination at the targeted site. Tofacilitate site-directed recombination a pair of TALENs was engineeredto introduce a double-strand break at a specific site within the L7locus. Vectors made according to Examples 1-3 (see Table 2) weretransformed into immature corn embryos. The vectors each comprised asequence of interest containing three expression cassettes: twoexpression cassettes for expression of each one of the TALEN pair and anexpression cassette containing a CP4-EPSPS transgene positioned betweena left homology arm and a right homology arm.

Approximately 3000 immature corn embryos were co-cultured withAgrobacterium containing one of the four vectors for 3 days, then movedto callus-induction medium containing 0.1 mM glyphosate as a selectionagent. Approximately 300 regenerated plants (RO events) were selectedfor each vector, transferred to plugs, and grown in a greenhouse.Genomic DNA was extracted from RO leaf tissue after one week ofgreenhouse growth, and individual plants were molecularly assessed withreal time PCR for (a) the presence of the transgene (CP4-EPSPS) copynumber, and (b) targeting sequence copy number. Individual plants thatscored “1” or “2” for transgene copy number (i.e., there are one or twocopies of the transgene inserted in the plant genome), or “0” or “1” fortargeting sequence copy number (i.e., there is a mutation at the genomictargeting sequence after TALEN enzyme cutting), were selected foradditional PCR analysis.

PCR with genomic DNA from of selected RO plants was used to identifyindividual plants comprising CP4-EPSPS cassette insertions at the L7target site. PCR primers were designed such that a product was onlyproduced when the CP4-EPSPS cassette inserted into the L7 targetedregion of the corn genome; one PCR primer was located in genomic DNAflanking the targeted region, and one PCR primer was located within theCP4-EPSPS cassette. Two sets of PCR primers were used, one positioned onthe 5′ end of the CP4-EPSPS cassette and one positioned on the 3′ end ofthe CP4-EPSPS cassette. FIG. 5 shows the positions of the primers usedto identify CP4-EPSPS cassette insertions in the L7 locus. The 5′ set ofPCR primers amplifies a 2676 bp product when the CP4-EPSPS cassette isinserted in the targeted L7 locus; no product is produced in wild typegenomic DNA or if the CP4-EPSPS cassette is not inserted into the L7target site. The 3′ set of PCR primers amplifies a 2282 bp product whenthe CP4-EPSPS is inserted in the targeted L7 locus; no product isproduced in wild type genomic DNA or if the CP4-EPSPS cassette is notinserted into the L7 target site. After PCR, the products were resolvedon an agarose gel to identify plants with the correct sized bands forboth the 5′ end and the 3′ end of the CP4-EPSPS cassette. The results ofthis analysis are shown in Table 2.

To further confirm TALEN-mediated site-directed integration, plants withat least one positive PCR result for site directed insertion of theCP4-EPSPS cassette were selected for Southern blot analysis. Genomic DNAextracted from the plant was digested with the restriction endonucleaseKpnI. Digestion with KpnI produces a 9.6 kb fragment when the CP4-EPSPScassette is integrated into the L7 target site (see FIG. 5 ). Digestionwith KpnI produces a 5.1 kb DNA fragment corresponding to the L7 genomiclocus, for example in the wild-type non-transformed plant or a plantwhere the CP4-EPSPS cassette has not integrated into the L7 locus (seeFIG. 5 ). The Southern blot was probed with a “left flank probe” asshown in FIG. 5 . The southern blot results showing a combination ofbands of a 9.6 kb (targeted integration) and a 5.1 kb (wildtype/non-targeted) indicated that transgenic plants were hemizygous forthe CP4-EPSPS cassette (i.e., the CP4-EPSPS cassette was only targetedto one corn chromosome). The southern blot results showing only a bandof 9.6 kb (targeted integration) indicated that transgenic plants werehomozygous for the CP4-EPSPS cassette (i.e., the CP4-EPSPS cassette wastargeted to both corn chromosomes). TALEN-mediated site-directedintegration (SDI) using Agrobacterium vectors comprising either two RBDNA sequences (vector C) or two RB DNA sequences and one LB DNA sequence(vector D) had superior efficacy compared to standard Agrobacteriumvectors comprising either one RB DNA sequence and one LB DNA sequence(vector A), or one RB DNA sequence (vector B). The data indicate theunexpected observation of an increased frequency of site-directedintegration by NHEJ or HR when the vector comprised two RB DNA sequencespositioned to generate essentially complementary T-strands. The datafurther indicated an increased number of events generated by HR when thevector comprised two RB DNA sequences positioned to generate essentiallycomplementary T-strands (see Table 2).

TABLE 2 Summary of border configurations and site-directed integration(SDI) frequencies. SDI positive events by # R0 Flank PCR Southern Totalplants Left Right L & R NHEJ Border embryos # screened Flank Flank Flankor Vector configuration transformed events by PCR Pos. Pos. Pos. HR HR A1 right border 2921 368 0 1 0 1 0 (SEQ ID NO: 4) 1 left border (SEQ IDNO: 19) B 1 right border 3177 228 68 2 2 2 1 1 (SEQ ID NO: 4) C 2 rightborders 3231 336 28 11 7 7 7 4 (SEQ ID NO: 4) (SEQ ID NO: 4 D 2 rightborders 3544 221 73 8 7 6 8 4 (SEQ ID NO: 4) (SEQ ID NO: 4) 1 leftborder (SEQ ID NO: 19) Pos., positive; L, left; R, right; NHEJ,non-homologous end-joining; HR, homologous recombination.

Example 10. Retargeting a Gene to a Pre-Existing Lox Site

A vector with two RB DNA sequences is used to facilitate site-directedintegration of a sequence of interest into a pre-existing recombinationsite (e.g. a lox site) in genomic DNA. The vector is created comprising,from 5′ to 3′, a first right RB DNA sequence (RB1); P-35S-crtB; a firstlox site; a gene; a first recombination site (optional); a marker geneto select for transformants; a second recombination site (optional); asecond lox site; P-DaMV-Cre; and a second RB DNA sequence (RB2). The RB1and RB2 are positioned in the vector to generate essentiallycomplementary T-strands. P-35S-crtB is a non-lethal, constitutivelyexpressed phytoene synthase expression cassette that inhibits shootelongation in cells where non-targeted recombination occurs. P-DaMV-Creis a constitutively expressed Cre-recombinase that promotesrecombination of the intervening DNA construct with the targeted genomiclox site.

Upon creation of a complementary strand of the intervening DNA duringAgrobacterium-mediated transformation, the lox sites of the interveningDNA recombine with a pre-existing genomic DNA lox site and insert intothe genomic DNA. A target after transformation and recombinationcontains only the regions of the intervening DNA construct between thetwo lox sites (in this example, a gene; a first recombination site; amarker gene; and a second recombination site). Optional recombinationsites are used to remove the marker gene in a future recombinationevent.

Example 11. Prolonged Co-Culturing During Agrobacterium-MediatedTransformation Increases Site-Directed Integration Frequency

For transformation of immature corn embryos, Agrobacterium containing avector is typically incubated with the immature corn embryos for 12-16hours. The unexpected observation detailed herein was that extendedco-culture of the Agrobacterium containing a vector with the immaturecorn embryos resulted in a higher percentage of targeted integrations ofthe sequence of interest contained in the vector while not significantlyaffecting transformation frequency.

Vector A (FIG. 4A, Example 1) was used for Agrobacterium-mediatedtransformation using standard protocols except that the co-culture timewas varied. About 3000 immature corn embryos were co-cultured for eachof 1 day, 2 days, or 3 days. After transformation, the transformantswere moved to callus-induction medium containing 0.1 mM glyphosate as aselection agent. RO plants surviving glyphosate selection weretransferred to plugs and grown in a greenhouse.

As shown in Table 3, the transformation frequency did not varysignificantly between co-culturing periods. The frequency of transformedplants surviving for molecular assessment (91.7% survival for 1 dayco-incubation; 91.3% survival for 2 day co-incubation; and 92.8%survival for 3 day co-incubation) shows that prolonged co-culturing didnot affect plant health after regeneration from callus. Furthermore, thepercentage transformation and the number of plants surviving formolecular assessment was similar for each of the 1 day, 2 day, and 3 dayco-culture periods.

TABLE 3 Summary of prolonged co-culture period forAgrobacterium-mediated transformation # of Plants Surviving for Co-Immature Molecular culturing Embryos Regenerated Assessment PercentagePeriod Used Plants (%) Transformed 1 day 3127 205 188 (91.7%) 6.0 2 day3138 321 293 (91.3%) 9.3 3 day 2920 263 244 (92.8%) 8.4

After one week of greenhouse growth, RO leaf tissue was sampled fromplants surviving for molecular assessment, and genomic DNA was extractedand assessed for the presence of and copy number of the transgenecassette (CP4-EPSPS), and for targeting sequence copy number with realtime PCR. Individual plants that scored “0” or “1” for targetingsequence copy number (i.e., there is a mutation at the genomic targetsite after TALEN enzyme cutting), or that scored a “1” or “2” fortransgene cassette copy number (i.e., there are one or two copies of thetransgene cassette inserted in the plant genome) were selected forfurther PCR analysis. The mutation rate of the targeted site is anindicator of how well the TALEN pair worked in plant cells. The mutationrate can be calculated by assaying the targeting sequence copy number inRO plants. As shown in Table 4, the mutation rate at the targeted siteincreased as the co-culturing period was prolonged.

TABLE 4 Mutation rate of each co-culturing period Co- R0 plants withPercentage of R0 plants culturing Surviving mutation at targeted withmutation at period R0 plants site targeted site 1 day 188  96 51.1 2 day293 162 55.3 3 day 244 149 61.1

The PCR protocol and primers described in Example 9 were used with thegenomic DNA from selected RO plants from the extended co-cultureprotocol to identify individual plants comprising targeted integrationof the CP4-EPSPS cassette. As described in Example 9, and illustrated inFIG. 5 , two sets of PCR primers were used to detect targetedintegration of the CP4-EPSPS cassette at the L7 locus. To further verifywhether the junction sequences at the targeted sites were perfect (e.g.,HR) or imperfect (e.g., NHEJ), the PCR products were sequenced. Table 5shows that the highest number of positive PCR results for either theleft flank, the right flank, or both the left and right flanks was withthe 3-day co-culture.

To further confirm the PCR results, RO plants with at least one positivePCR result were selected for Southern blot analysis, as detailed inExample 9. Genomic DNA extracted from the plant was digested with therestriction endonuclease KpnI, and the southern blots were probed with aleft-flank probe (FIG. 5 ). For each co-culturing period, PCR andSouthern blot results detecting targeted integration of the CP4-EPSPScassette at the L7 locus are summarized in Table 5. The data indicatethe unexpected observation of an increased frequency of site-directedintegration by NHEJ or HR with the 3-day co-culture. This findingdemonstrates that Agrobacterium-mediated transformation is moreefficient at inducing site-directed integrations in immature cornembryos with prolonged co-culturing periods.

TABLE 5 Summary of left and right flank PCR, southern results andpercentage of SDI and HR frequencies in co-culture experiment. PCR RightLeft Right and Southern Co- Embryos Flank Flank Left Targeted Targetedculturing trans- # of Pos- Pos- Flank (NHEJ (HR period formed eventsitive itive Positive or HR) only) 1 day 3127 205 1 0 0 1 0 2 day 3138321 0 0 0 0 0 3 day 2920 263 5 6 5 6 3 NHEJ, non-homologous end joining;HR, homologous recombination

Example 12: Increased Frequency of Site-Directed Integration of aSequence of Interest Flanked by RBs and Optional LBs

A site within the L7 locus, described in Example 9, was selected forsite-directed integration of a transgene cassette. An expressioncassette encoding CP4-EPSPS (CP4-EPSPS), which confers tissue-restrictedtolerance to the herbicide glyphosate, was chosen as the cargo forsite-specific integration at the L7 locus. To facilitate site-directedintegration, a pair of TALENs was engineered to introduce adouble-strand break at a specific site within the L7 locus.

A control vector (Vector 6A) was created for Agrobacterium-mediatedtransformation (see FIG. 6A). The control vector comprised a firstsequence of interest comprising an expression cassette encodingCP4-EPSPS positioned between a first RB DNA sequence (RB1) and a firstLB DNA sequence (LB1) such that T-strand synthesis initiated at RB1 andterminated at LB1. The vector further comprised a second sequence ofinterest comprising two expression cassettes each encoding half of aTALEN pair positioned between a second RB DNA sequence (RB2) and asecond LB DNA sequence (LB2) such that T-strand synthesis initiated atRB2 and terminated at LB2. TALENs were obtained from Life Technologies.

A second vector (Vector 6B) was created for Agrobacterium-mediatedtransformation (see FIG. 6B). This vector comprised a first sequence ofinterest comprising an expression cassette encoding CP4-EPSPS positionedbetween two RB DNA sequences and two LB DNA sequences. The vectorfurther comprised a second sequence of interest comprising twoexpression cassettes each encoding half of a TALEN pair. The vectorconfiguration comprised a first RB DNA sequence (RB1) paired with afirst LB DNA sequence (LB1) which were positioned in the vector toinitiate (RB1) and terminate (LB1) synthesis of a first T-strandcomprising CP4-EPSPS in an anti-sense orientation from the 5′ to 3′ endof the first T-strand; and the second RB DNA sequence (RB2) was pairedwith a second LB DNA sequence (LB2) which were positioned in the vectorto initiate synthesis of a second T-strand such that CP4-EPSPS was insense orientation from the 5′ to 3′ end of the second T-strand.Furthermore, RB1 was also positioned such that read through of the LB1would result in the synthesis of a T-strand comprising both theCP4-EPSPS cassette and the two TALEN cassettes. The T-strands resultingfrom initiation at RB1 and RB2 were essentially complementary to eachother in at least a portion of the first sequence of interest.

Approximately 3000 to 5000 immature corn embryos were co-cultured withAgrobacterium containing either the control Vector 6A or Vector 6B for 3days, then moved to callus-induction medium containing 0.1 mM glyphosateas a selection agent. One hundred eighty regenerated plants (RO events)were selected for Vector 6A and 371 regenerated plants were selected forVector 6B, transferred to plugs, and grown in a greenhouse.

To confirm TALEN mediated site directed integration, genomic DNA wasisolated from selected RO plants and PCR assays were carried out toidentify individual plants comprising CP4-EPSPS cassette insertions atthe L7 target site. PCR primers were designed such that a product wasonly produced when the CP4-EPSPS cassette inserted into the L7 targetregion of the corn genome. PCR protocol and primers described in Example9, and illustrated in FIG. 5 , were used to identify individual plantscomprising targeted integration of the CP4-EPSPS cassette. FollowingPCR, the PCR products were resolved on agarose gels to identify plantswith the correct sized bands for both the 5′ end and the 3′ end of theCP4-EPSPS cassette. As tabulated in Table 6, for the control Vector 6A:ofthe 180 RO plants that were screened by PCR, none were positive forthe left flank and 2 were positive for the right flank. For vector 6B,of the 371 RO plants that were screened by PCR, 2 were positive for theleft flank and 10 were positive for the right flank. The data indicatedan increased frequency of site directed integration when the vectorcomprises two RB DNA sequences positioned to generate essentiallycomplementary T-DNA strands containing the cargo sequence to be inserted(Vector 6B).

TABLE 6 Summary of border configurations and site-directed integrationfrequencies. Flank PCR Total Left and embryos Left Right Right Bordertrans- # Flank Flank Flank Vector configuration formed events PositivePositive Positive 6A 2 right borders 3125 180 0 2 0 (SEQ ID NO: 4) (SEQID NO: 12) 2 left borders (SEQ ID NO: 19) (SEQ ID NO: 17) 6B 2 rightborders 5445 371 2 10 0 (SEQ ID NO: 4) (SEQ ID NO: 12) 2 left borders(SEQ ID NO: 19) (SEQ ID NO: 17)

Example 13: Increased Frequency of Site-Directed Integration of aSequence of Interest Flanked by RBs and No LBs

A site within the L7 locus, described in Example 9, was selected forsite-directed integration of a transgene cassette encoding a CP4-EPSPS(CP4-EPSPS), which confers tolerance to the herbicide glyphosate. Tofacilitate site-directed integration of the cassette, a pair of TALENswas engineered to introduce a double-strand break at a specific sitewithin the L7 locus.

A first vector (Vector 7A) was created for Agrobacterium-mediatedtransformation. For illustrative purposes, the vector configuration ispresented in FIG. 7A. The vector comprised a first sequence of interestcomprising an expression cassette encoding CP4-EPSPS with a TALEN targetsite (TS) positioned 5′ to the cassette. Additionally, the firstsequence of interest was flanked by a first RB DNA sequence (RB1) and asecond RB DNA sequence (RB2) positioned such that T-strand synthesisinitiated at both RB1 and RB2 and the resulting T-strands wereessentially complementary to each other in at least a portion of thefirst sequence of interest. The vector further comprised a secondsequence of interest comprising two expression cassettes each encodinghalf of a TALEN pair, where the second sequence of interest waspositioned adjacent to a third RB DNA sequence (RB3) so as to initiatesynthesis of a third T-strand that comprised the two TALEN cassettes.TALENs were obtained from Life Technologies

A second vector (Vector 7B) was created for Agrobacterium-mediatedtransformation. For illustrative purposes, the vector configuration ispresented in FIG. 7B. The vector comprised a first sequence of interestcomprising an expression cassette encoding CP4-EPSPS flanked by TALENtarget sites (TS). Additionally, the first sequence of interest waspositioned between a first RB DNA sequence (RB1) and a second RB DNAsequence (RB2) such that T-strand synthesis initiated at both RB1 andRB2 and the resulting T-strands were essentially complementary to eachother in at least a portion of the first sequence of interest. Thevector further comprised a second sequence of interest comprising twoexpression cassettes each encoding half of a TALEN pair positionedadjacent to a third RB DNA sequence (RB3) so as to initiate synthesis ofa third T-strand that comprised the two TALEN cassettes. TALENs wereobtained from Life Technologies.

A third vector (Vector 7C) was created for Agrobacterium-mediatedtransformation. For illustrative purposes, the vector configuration ispresented in FIG. 7C. The vector comprised a first sequence of interestcomprising an expression cassette encoding CP4-EPSPS positioned betweena left homology arm and a right homology arm. Additionally, the firstsequence of interest was positioned between a first RB DNA sequence(RB1) and a second RB DNA sequence (RB2) such that T-strand synthesisinitiated at both RB1 and RB2 and the resulting T-strands wereessentially complementary to each other in at least a portion of thefirst sequence of interest. The vector further comprised a secondsequence of interest comprising two expression cassettes each encodinghalf of a TALEN pair positioned adjacent to a third RB DNA sequence(RB3) so as to initiate synthesis of a third T-strand that comprised thetwo TALEN cassettes. TALENs were obtained from Life Technologies.

Approximately 4000 to 5000 immature corn embryos were co-cultured withAgrobacterium containing one of the three vectors for 3 days, then movedto callus-induction medium containing 0.1 mM glyphosate as a selectionagent. Approximately 200 regenerated plants (RO events) were selectedfor each vector, transferred to plugs, and grown in a greenhouse.

To confirm TALEN mediated site directed integration, genomic DNA wasisolated from selected RO plants and PCR assays were carried out toidentify individual plants comprising CP4-EPSPS cassette insertions atthe L7 target site. PCR primers were designed such that a product wasonly produced when the CP4-EPSPS cassette inserted into the targeted L7region of the corn genome. PCR protocol and primers described in Example9, and illustrated in FIG. 5 , were used to identify individual plantscomprising targeted integration of the CP4-EPSPS cassette. After PCR,the products were resolved on agarose gels to identify plants with thecorrect sized bands for both the 5′ end and the 3′ end of the CP4-EPSPScassette. As tabulated in Table 7, for Vector 7A: of the 226 RO plantsthat were screened by PCR, one was positive for the left flank and 4were positive for the right flank. For Vector 7B, of the 292 RO plantsthat were screened by PCR, 2 were positive for the left flank and 26were positive for the right flank. One plant was positive for both rightand left PCR. For Vector 7C, of the 241 RO plants that were screened byPCR, 7 were positive for the left flank and 7 were positive for theright flank. Four plants were positive for both right and left flank PCRproducts.

The data indicate that TALEN-mediated site-directed integration (SDI)using an Agrobacterium vector comprising two TALEN target sites and twoRB DNA sequences flanking a cargo sequence of interest (Vector 7B) hadsuperior efficacy compared to an Agrobacterium vector comprising only 1target site and two RB DNA sequences flanking the cargo sequence (Vector7A). The data further indicate an increased number of full integrationevents generated by HR when the vector comprised a cargo sequenceflanked by homology arms and two RB DNA sequences (Vector 7C).

TABLE 7 Summary of border configurations and site-directed integrationfrequencies. Flank PCR Total Left and embryos Left Right Right Bordertrans- # Flank Flank Flank Vector configuration formed events PositivePositive Positive 7A 3 right borders 5235 226 1 4 0 (SEQ ID NO: 4) (SEQID NO: 4) (SEQ ID NO: 12) 7B 3 right borders 4709 292 2 26 1 (SEQ ID NO:4) (SEQ ID NO: 4) (SEQ ID NO: 12) 7C 3 right borders 4059 241 7 7 4 (SEQID NO: 4) (SEQ ID NO: 4) (SEQ ID NO: 12)

Example 14. Co-Culturing Time During Agrobacterium-MediatedTransformation Influences Site-Directed Integration Frequency

For transformation of immature corn embryos, Agrobacterium comprising atransformation vector is typically incubated with immature corn embryosfor 12-16 hours. The unexpected observation detailed herein was thatprolonging the co-culture time of the Agrobacterium comprising a vectorwith immature corn embryos to three days resulted in a higher number ofevents that had targeted integration of the sequence of interestcontained in the vector.

A vector depicted in FIG. 4A and Example 1 was used forAgrobacterium-mediated transformation using standard protocols exceptthat the co-culture time was varied. Explants were co-cultured withAgrobacterium for 1, 3, 5, or 7 days. Transformation of immature embryoswas conducted on eight different experimental dates. One and three dayco-culture treatments were conducted on alternating dates (a total of 4experimental treatment dates each), and an average of 1086 and 1132explants were treated per experiment, respectively. Five and seven daytreatments were conducted on each of the eight experimental dates, withan average of 558 and 556 explants treated per experiment, respectively.After transformation, the transformants were moved to callus-inductionmedium containing 0.1 mM glyphosate as a selection agent. RO plantssurviving glyphosate selection were transferred to plugs and grown in agreenhouse. The number of stable herbicide tolerant events generated perexperimental treatment was divided by the number of explants treated todetermine transformation frequencies. PCR protocol and primers describedin Example 9 were used with the genomic DNA from RO plants from theextended co-culture protocol to identify individual plants comprisingtargeted integration of the CP4-EPSPS cassette. As described in Example9, and illustrated in FIG. 5 , two sets of PCR primers were used todetect targeted integration of the CP4-EPSPS cassette at the L7 locus.The number of site directed integration events (determined by positiveflank PCR results for either or both flanks) detected per experimentaltreatment was divided by the number of explants recovered to estimatethe SDI frequencies. The data were analyzed using a mixed linear model.Table 8 shows that the highest number of positive PCR results for eitherthe left flank, the right flank, or both the left and right flanks waswith the 3-day co-culture.

TABLE 8 Summary of left and right flank PCR, transformation frequencyand percentage of SDI in co-culture experiment. PCR Co- Embryos TFN Leftor Right Right and culturing trans- # of Freq Flank Pos. SDI Left Flankperiod formed events (%) (SDI events) (%) Pos. 1 day 4345 387  8.9 8 2.11 3 day 4530 527 11.6 21 4.0 3 5 day 4478 222  5.0 10 4.5 1 7 day 4455126  2.8 6 4.8 0 TFN, Transformation; Pos., positive; SDI, site-directedintegration

1. A method of providing a sequence of interest to the genome of a plantcell, comprising contacting the plant cell with a Rhizobiales cellcapable of transforming the plant cell, wherein the Rhizobiales cellcomprises at least one vector capable of forming two T-strands that areessentially complementary in at least a portion of the T-strands, a.wherein the at least one vector comprises a first right border DNAsequence (RB1), a second right border DNA sequence (RB2), and at leastone sequence of interest, and wherein the RB1 is positioned in thevector to initiate synthesis of a first T-strand such that the sequenceof interest is in the sense orientation from the 5′ to 3′ end of thefirst T-strand; and the RB2 is positioned in the vector to initiatesynthesis of a second T-strand such that the sequence of interest is inthe anti-sense orientation relative to the sequence of interest in thefirst T-strand, and wherein the two T-strands resulting from initiationat RB1 and RB2 are essentially complementary in at least a portion ofthe sequence of interest; or b. wherein the at least one vectorcomprises a RB1, a RB2, a sequence of interest, a first left border DNAsequence (LB1) and a second left border DNA sequence (LB2), wherein thevector is configured such that the RB1 is paired with the LB1 which arepositioned in the vector to initiate (RB1) and terminate (LB1) synthesisof a first T-strand such that the sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the RB2 ispaired with the LB2 which are positioned in the vector to initiate (RB2)and terminate (LB2) synthesis of a second T-strand such that thesequence of interest is in an anti-sense orientation relative to thesequence of interest in the first T-strand, and wherein the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest; or c.wherein the vector comprises a first sequence of interest and a secondsequence of interest, wherein the first sequence of interest isessentially identical to the second sequence of interest; wherein thevector further comprises a RB1 and a LB1 which are positioned in thevector to initiate (RB1) and terminate (LB1) synthesis of a firstT-strand such that the first sequence of interest is in the senseorientation from the 5′ to 3′ end of the first T-strand; and the vectorfurther comprises a RB2 and a LB2 which are positioned in the vector toinitiate (RB2) and terminate (LB2) synthesis of a second T-strand suchthat the second sequence of interest is in an anti-sense orientationrelative to the first sequence of interest in the first T-strand, andwherein the two T-strands resulting from initiation at RB1 and RB2 areessentially complementary in at least a portion of the first sequence ofinterest and the second sequence of interest; or d. wherein theRhizobiales cell comprises at least a first vector and a second vector,wherein each vector comprises essentially identical sequences ofinterest, and wherein the first vector comprises a RB1 and a LB1 whichare positioned in the first vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andwherein the second vector comprises a RB2 and a LB2 which are positionedin the second vector to initiate (RB2) and terminate (LB2) synthesis ofa second T-strand such that the sequence of interest is in an anti-senseorientation relative to the sequence of interest in the first T-strand,and wherein the two T-strands resulting from initiation at RB1 and RB2are essentially complementary in at least a portion of the sequence ofinterest; or e. wherein the at least one vector comprises a firstsequence of interest, a second sequence of interest different from thefirst sequence of interest, at least two RB DNA sequences, and one ormore optional LB DNA sequences, wherein the first RB DNA sequence (RB1)and a first LB DNA sequence (LB1) are positioned in the vector toinitiate (RB1) and terminate (LB1) synthesis of the first T-strand suchthat the first sequence of interest is in the sense orientation from the5′ to 3′ end of the first T-strand; and the vector configuration furthercomprises a second RB DNA sequence (RB2) and a second LB DNA sequence(LB2) which are positioned in the vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the first sequence ofinterest is in an anti-sense orientation from the 5′ to 3′ end of thesecond T-strand, wherein the two T-strands resulting from initiation atRB1 and RB2 are essentially complementary to each other in at least aportion of the first sequence of interest, and wherein the vectorconfiguration further comprises a third RB DNA sequence (RB3) and athird LB DNA sequence (LB3) which are positioned in the vector toinitiate (RB3) and terminate (LB3) synthesis of a third T-strand suchthat the second sequence of interest is in the sense orientation fromthe 5′ to 3′ end of the third T-strand; and the vector configurationfurther comprises a fourth RB DNA sequence (RB4) and a fourth LB DNAsequence (LB4) which are positioned in the vector to initiate (RB4) andterminate (LB4) synthesis of a fourth T-strand such that the secondsequence of interest is in an anti-sense orientation from the 5′ to 3′end of the fourth T-strand, and the two T-strands resulting frominitiation at RB3 and RB4 are essentially complementary to each other inat least a portion of the second sequence of interest.
 2. (canceled) 3.The method of claim 1, wherein a. the RB1 and the RB2 are essentiallyhomologous, and the LB1 and the LB2 are essentially homologous; b. theRB1 and the RB2 are essentially homologous, and the LB1 and the LB2 arenot essentially homologous; c. the RB1 and the RB2 are not essentiallyhomologous, and the LB1 and the LB2 are essentially homologous; or d.the RB1 and the RB2 are not essentially homologous, and the LB1 and theLB2 are not essentially homologous.
 4. The method of claim 1, wherein a.at least one of the RB1 and RB2 comprise an Agrobacterium Ti plasmidright border consensus DNA sequence; b. at least one of the RB1 and RB2comprise an Agrobacterium Ti plasmid right border consensus DNA sequenceselected from SEQ ID NO: 21 and SEQ ID NO: 22; c. at least one of theRB1 and RB2 comprise a sequence selected from SEQ ID NOs: 1-13; or d. atleast one of the RB1 and RB2 comprise a sequence at least 80% identicalto a sequence selected from SEQ ID NO:4 and SEQ ID NO:12.
 5. The methodof claim 1, wherein a. at least one of the LB1 and LB2 comprise anAgrobacterium Ti plasmid left border consensus DNA sequence; b. at leastone of the LB1 and LB2 comprise an Agrobacterium Ti plasmid left borderconsensus DNA sequence selected from SEQ ID NO: 23 and SEQ ID NO: 24; c.at least one of the LB1 and LB2 comprise a sequence selected from SEQ IDNOs: 14-20; or d. at least one of the LB1 and LB2 comprise a sequence atleast 80% identical to SEQ ID NO:19.
 6. (canceled)
 7. The method ofclaim 1, wherein the sequence of interest comprises one or moresequences selected from: a gene, a portion of a gene, an intergenicsequence, an enhancer, a promoter, an intron, an exon, a sequenceencoding a transcription termination sequence, a sequence encoding achloroplast targeting peptide, a sequence encoding a mitochondrialtargeting peptide, an insulator sequence, a sequence encoding ananti-sense RNA construct, a sequence encoding a protein, a sequenceencoding non-protein-coding RNA (npcRNA), a sequence encoding arecombinase, a sequence encoding a recombinase recognition site, alanding pad, an editing template, an expression cassette, a stack of twoor more expression cassettes encoding transgenes, a sequence encoding asite-specific enzyme, a sequence encoding a site-specific enzyme targetsite, a sequence encoding a selection marker, a sequence encoding a cellfactor that functions to increase DNA repair, a sequence comprising alinker or a spacer, a sequence comprising one or more restriction enzymesites, a sequence for templated genome editing, and any combinationthereof.
 8. The method of claim 6, wherein the sequence of interestfurther comprises at least one homology arm DNA sequence.
 9. The methodof claim 8, wherein the sequence of interest comprises both a lefthomology arm DNA sequence and a right homology arm DNA sequence.
 10. Themethod of claim 8, wherein the at least one homology arm DNA sequencecomprises a sequence that is at least 80% identical to a target sequencein the plant genome. 11.-12. (canceled)
 13. The method of claim 7,wherein the site-specific enzyme is selected from a group consisting ofan endonuclease, a recombinase, and a transposase. 14.-16. (canceled)17. The method of claim 7, wherein the sequence of interest comprises asequence encoding a protein involved in DNA repair, wherein the proteinis selected from the group comprising a vir gene from the Ti plasmid,Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combinationthereof.
 18. The method of claim 1, wherein at least part of thesequence of interest is integrated into the plant genome via homologousrecombination or via non-homologous end joining, wherein the integrationof at least part of the sequence of interest results in a pointmutation, an insertion, a deletion, an inversion, increasedtranscription of an endogenous locus, decreased transcription of anendogenous locus, altered protein activity, altered RNAi products,altered RNAi target sites, altered RNAi pathway activity, increasedtranscription of the sequence of interest, decreased transcription ofthe integrated sequence of interest, or any combination thereof.
 19. Amethod of providing a sequence of interest to the genome of a plantcell, comprising contacting the plant cell with a Rhizobiales cellcapable of transforming the plant cell, wherein the Rhizobiales cellcomprises at least one vector comprising a right border DNA sequence(RB) and a left border DNA sequence (LB) and wherein the vectorcomprises between the RB and LB: (i) a first sequence of interest in asense orientation relative to the RB, (ii) a spacer, and (iii) a secondsequence of interest in an anti-sense orientation relative to the RB,wherein the first sequence of interest and second sequence of interestare essentially complementary and after synthesis of the T-strand annealto form a double-stranded DNA.
 20. The method of claim 19, wherein thefirst sequence of interest further comprises a first left homology armDNA sequence and a first right homology arm DNA sequence, and the secondsequence of interest further comprises a second left homology arm DNAsequence and a second right homology arm DNA sequence.
 21. The method ofclaim 1, wherein the Rhizobiales cell is selected from an Agrobacteriumspp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp.,a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp. 22.(canceled)
 23. The method of claim 1, wherein the plant cell is selectedfrom the group consisting of a corn cell, a soybean cell, a canola cell,a cotton cell, a wheat cell, or a sugarcane cell. 24.-45. (canceled) 46.A method of providing a sequence of interest to a plant genome,comprising contacting at least one plant cell on a co-culture medium forat least 2 days, with at least one Rhizobiales cell capable oftransforming the plant cell, wherein the Rhizobiales cell comprises atleast one vector capable of forming two essentially complementaryT-strands, wherein, a. the at least one vector comprises a first rightborder (RB1) DNA sequence, a second right border DNA sequence (RB2), andat least one sequence of interest, and wherein the RB1 is positioned inthe vector to initiate synthesis of a first T-strand such that thesequence of interest is in the sense orientation from the 5′ to 3′ endof the first T-strand; and the RB2 is positioned in the vector toinitiate synthesis of a second T-strand such that the sequence ofinterest is in the anti-sense orientation relative to the sequence ofinterest in the first T-strand, and wherein the sequence of interest inthe two T-strands resulting from initiation at RB1 and RB2 areessentially complementary in at least a portion of the sequence ofinterest; or b. the at least one vector disclosed herein comprises aRB1, a RB2, and a sequence of interest, and further comprises a firstleft border DNA sequence (LB1) and a second left border DNA sequence(LB2), and wherein the vector is configured such that the RB1 is pairedwith the LB1 which are positioned in the vector to initiate (RB1) andterminate (LB1) synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the RB2 is paired with the LB2 which are positioned in thevector to initiate (RB2) and terminate (LB2) synthesis of a secondT-strand such that the sequence of interest is in an anti-senseorientation relative to the sequence of interest in the first T-strand,and wherein the sequence of interest in the two T-strands resulting frominitiation at RB1 and RB2 are essentially complementary in at least aportion of the sequence of interest; or c. the at least one vectorcomprises a first sequence of interest and a second sequence ofinterest, where the first sequence of interest is essentially identicalto the second sequence of interest; and the vector configuration furthercomprises a RB1 with aLB1 which are positioned in the vector to initiate(RB1) and terminate (LB1) synthesis of a first T-strand such that thefirst sequence of interest is in the sense orientation from the 5′ to 3′end of the first T-strand; and the vector further comprises a RB2 and aLB2 which are positioned in the vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the second sequence ofinterest is in an anti-sense orientation relative to the first sequenceof interest in the first T-strand, and wherein the sequence of interestin the two T-strands resulting from initiation at RB1 and RB2 areessentially complementary in at least a portion of the first sequence ofinterest and the second sequence of interest; or d. the Rhizobiales cellcomprises at least a first vector and a second vector, wherein eachvector comprises essentially identical sequences of interest, andwherein the first vector configuration comprises a RB1 and a LB1 whichare positioned in the first vector to initiate (RB1) and terminate (LB1)synthesis of a first T-strand such that the sequence of interest is inthe sense orientation from the 5′ to 3′ end of the first T-strand; andwherein the second vector configuration comprises a RB2 and a LB2 whichare positioned in the second vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the sequence of interestis in an anti-sense orientation relative to the sequence of interest inthe first T-strand, and wherein the sequence of interest in the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest; or e.the at least one vector comprises a first sequence of interest, a secondsequence of interest different from the first sequence of interest, atleast two RB DNA sequences, and one or more optional LB DNA sequences,wherein the first RB DNA sequence (RB1) and a first LB DNA sequence(LB1) are positioned in the vector to initiate (RB1) and terminate (LB1)synthesis of the first T-strand such that the first sequence of interestis in the sense orientation from the 5′ to 3′ end of the first T-strand;and the vector configuration further comprises a second RB DNA sequence(RB2) and a second LB DNA sequence (LB2) which are positioned in thevector to initiate (RB2) and terminate (LB2) synthesis of a secondT-strand such that the first sequence of interest is in an anti-senseorientation from the 5′ to 3′ end of the second T-strand, wherein thetwo T-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary to each other in at least a portion of the first sequenceof interest, and wherein the vector configuration further comprises athird RB DNA sequence (RB3) and a third LB DNA sequence (LB3) which arepositioned in the vector to initiate (RB3) and terminate (LB3) synthesisof a third T-strand such that the second sequence of interest is in thesense orientation from the 5′ to 3′ end of the third T-strand; and thevector configuration further comprises a fourth RB DNA sequence (RB4)and a fourth LB DNA sequence (LB4) which are positioned in the vector toinitiate (RB4) and terminate (LB4) synthesis of a fourth T-strand suchthat the second sequence of interest is in an anti-sense orientationfrom the 5′ to 3′ end of the fourth T-strand, and the two T-strandsresulting from initiation at RB3 and RB4 are essentially complementaryto each other in at least a portion of the second sequence of interest;or f. the at least one Rhizobiales cell comprises a first Rhizobialescell and a second Rhizobiales cell, wherein each Rhizobiales cellcontains at least one of two vectors, wherein each vector comprises anessentially identical sequence of interest, and where the first vectorcomprises a RB1, and wherein the RB1 is positioned in the vector toinitiate synthesis of a first T-strand such that the sequence ofinterest is in the sense orientation from the 5′ to 3′ end of the firstT-strand; and the second vector comprises a RB2 which is positioned inthe vector to initiate synthesis of a second T-strand such that thesequence of interest is in the anti-sense orientation relative to thesequence of interest in the first T-strand, and wherein the twoT-strands resulting from initiation at RB1 and RB2 are essentiallycomplementary in at least a portion of the sequence of interest; or g.the at least one Rhizobiales cell comprises a first Rhizobiales cell anda second Rhizobiales cell, wherein each Rhizobiales cell contains atleast one of two vectors, wherein each vector comprises an essentiallyidentical sequence of interest, and where the first vector comprises aRB1 and a LB1 which are positioned in the first vector to initiate (RB1)and terminate (LB1) synthesis of a first T-strand such that the sequenceof interest is in the sense orientation from the 5′ to 3′ end of thefirst T-strand; and the second vector comprises a RB2 and a LB2 whichare positioned in the second vector to initiate (RB2) and terminate(LB2) synthesis of a second T-strand such that the sequence of interestis in an anti-sense orientation from the 5′ to 3′ end of the secondT-strand, and wherein the sequence of interest in the two T-strandsresulting from initiation at RB1 and RB2 are essentially complementaryto each other.
 47. The method of claim 46, wherein the contactingcomprises co-culturing the plant cell with the Rhizobiales cell for atleast 3 days.
 48. The method of claim 46, wherein the Rhizobiales cellis selected from the group consisting of an Agrobacterium spp. cell, aBradyrhizobium spp. cell, a Mesorhizobium spp. cell, an Ochrobactrumspp. cell, a Phyllobacterium spp. cell, a Rhizobium spp. cell, and aSinorhizobium spp. cell.
 49. (canceled)
 50. The method of claim 46,wherein the plant cell is selected from the group consisting of a cornimmature embryo cell, a corn mature embryo cell, a corn seed cell, asoybean immature embryo cell, a soybean mature embryo cell, a soybeanseed cell, a canola immature embryo cell, a canola mature embryo cell, acanola seed cell, a cotton immature embryo cell, a cotton mature embryocell, a cotton seed cell, a wheat immature embryo cell, a wheat matureembryo cell, a wheat seed cell, a sugarcane immature embryo cell, asugarcane mature embryo cell, and a sugarcane seed cell.
 51. (canceled)